U.S. patent application number 17/500107 was filed with the patent office on 2022-03-31 for nutritional compositions containing butyrate and uses thereof.
This patent application is currently assigned to MEAD JOHNSON NUTRITION COMPANY. The applicant listed for this patent is MEAD JOHNSON NUTRITION COMPANY. Invention is credited to Gabriele Gross, Teartse Tim Lambers, Shay Phillips, Marieke H. Schoemaker, Eric A.F. van Tol.
Application Number | 20220095666 17/500107 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-31 |
United States Patent
Application |
20220095666 |
Kind Code |
A1 |
Schoemaker; Marieke H. ; et
al. |
March 31, 2022 |
NUTRITIONAL COMPOSITIONS CONTAINING BUTYRATE AND USES THEREOF
Abstract
Provided are nutritional compositions containing dietary
butyrate. Further disclosed are methods for reducing the incidence
of obesity and metabolic syndrome in a target subject via
administering the nutritional composition containing dietary
butyrate to the target subject. Further provided are methods for
improving adipose tissue functioning in a target subject.
Inventors: |
Schoemaker; Marieke H.;
(Rhenen, NL) ; Lambers; Teartse Tim; (Nijmegen,
NL) ; Gross; Gabriele; (Nijmegen, NL) ; van
Tol; Eric A.F.; (Arnhem, NL) ; Phillips; Shay;
(Oakland City, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEAD JOHNSON NUTRITION COMPANY |
Evansville |
IN |
US |
|
|
Assignee: |
MEAD JOHNSON NUTRITION
COMPANY
Evansville
IN
|
Appl. No.: |
17/500107 |
Filed: |
October 13, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15376330 |
Dec 12, 2016 |
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17500107 |
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International
Class: |
A23L 33/00 20060101
A23L033/00; A23L 33/135 20060101 A23L033/135; A23L 33/12 20060101
A23L033/12; A23L 33/10 20060101 A23L033/10; A23L 33/17 20060101
A23L033/17; A23L 33/15 20060101 A23L033/15; A23L 33/155 20060101
A23L033/155; A23L 33/16 20060101 A23L033/16; A61K 31/19 20060101
A61K031/19; A61K 31/716 20060101 A61K031/716; A61K 31/59 20060101
A61K031/59; A61K 38/01 20060101 A61K038/01; A61P 3/08 20060101
A61P003/08; A61P 3/02 20060101 A61P003/02; A61K 35/747 20060101
A61K035/747; A61K 45/06 20060101 A61K045/06; A61K 33/26 20060101
A61K033/26; A61P 3/00 20060101 A61P003/00; A61P 3/04 20060101
A61P003/04; A61K 31/047 20060101 A61K031/047; A61K 31/202 20060101
A61K031/202; A23L 33/18 20060101 A23L033/18; A61K 33/30 20060101
A61K033/30 |
Claims
1-21. (canceled)
22. A staged feeding regimen for an infant or child, comprising the
steps of: A) providing a first nutritional composition comprising
from about 4 .mu.g/mL to about 36 .mu.g/mL of dietary butyrate to
the infant from birth to about 3 months of age; B) providing a
second nutritional composition comprising from about 6 .mu.g/mL to
about 33 .mu.g/mL of butyrate to the infant from 3 months of age to
about 6 months of age; and C) providing a third nutritional
composition comprising from about 8 .mu.g/mL to about 40 .mu.g/mL
of butyrate to the infant from 6 months of age to about 12 months
of age.
23. The staged feeding regimen according to claim 22, wherein the
first nutritional composition comprises from about 7 .mu.g/mL to
about 22 .mu.g/mL of dietary butyrate; the second nutritional
composition comprises from about 17 .mu.g/mL to about 24 .mu.g/mL
of butyrate; and the third nutritional composition comprises from
about 17 .mu.g/mL to about 22 .mu.g/mL of butyrate.
24. The staged feeding regimen of claim 22, wherein the first
nutritional composition further comprises at least one of the
following selected from the group consisting of: i) up to about 0.2
mg/100 kcal of iron; ii) up to about 0.7 mg/100 kcal of zinc; iii)
up to about 2 g/100 kcal of protein; iv) a protein source
comprising a whey:casein ratio of at least about 72:28; v) up to 35
mg/100 kcal of inositol; and vii) from about 1.8 .mu.g/100 kcal to
about 2.0 .mu.g/100 kcal of vitamin D.
25. The staged feeding regimen of claim 22, wherein the second
nutritional composition further comprises at least one of the
following selected from the group consisting of: i) from about 0.3
mg/100 kcal to about 0.6 mg/100 kcal of iron; ii) up to about 0.9
mg/100 kcal of zinc; iii) up to about 1.9 g/100 kcal of protein;
iv) a protein source comprising a whey:casein ratio of about 65:44
to about 68:32; v) up to 25 mg/100 kcal of inositol; vi) up to 9.0
mg/100 kcal of butyrate; and vii) from about 1.8 .mu.g/100 kcal to
about 2.0 .mu.g/100 kcal of vitamin D.
26. The staged feeding regimen of claim 22, wherein the third
nutritional composition further comprises at least one of the
following selected from the group consisting of: i) from about 0.3
mg/100 kcal to about 1.2 mg/100 kcal of iron; ii) up to about 1
mg/100 kcal of zinc; iii) up to about 1.5 g/100 kcal of protein;
iv) a protein source comprising a whey:casein ratio of about 45:55
to about 56:44; v) up to 20 mg/100 kcal of inositol; vi) up to 9.0
mg/100 kcal of butyrate; and vii) from about 1.8 .mu.g/100 kcal to
about 2.0 .mu.g/100 kcal of vitamin D.
27. The staged feeding regimen of claim 22, wherein the first
nutritional composition, second nutritional composition, and third
nutritional composition each further comprise docosahexaenoic
acid.
28. The staged feeding regimen of claim 22, wherein the first
nutritional composition, second nutritional composition, and third
nutritional composition each further comprise a carbohydrate
source, a fat source, and a protein source.
29. The staged feeding regimen of claim 22, wherein the first
nutritional composition, second nutritional composition, and third
nutritional composition each further comprise prebiotics.
30. The staged feeding regimen of claim 22, wherein the first
nutritional composition, second nutritional composition, and third
nutritional composition each further comprise Lactobacillus
rhamnosus GG.
31. The staged feeding regimen of claim 22, wherein the first
nutritional composition, second nutritional composition, and third
nutritional composition each further comprise .beta.-glucan.
32. A staged feeding regimen for an infant or child, comprising the
steps of: A) providing a first nutritional composition comprising:
up to about 0.45 mg/100 kcal of iron; up to about 0.7 mg/100 kcal
of zinc; up to about 2 g/100 kcal of protein; a whey:casein ratio
of at least 72:28; up to 35 mg/100 kcal of inositol; up to 9.0
mg/100 kcal of butyrate; and/or from about 1.8 .mu.g/100 kcal to
about 2.0 .mu.g/100 kcal of vitamin D; B) providing a second
nutritional composition comprising: from about 0.3 mg/100 kcal to
about 0.6 mg/100 kcal of iron; up to about 0.9 mg/100 kcal of zinc;
up to about 1.9 g/100 kcal of protein; a whey:casein ratio of
between 56:44 and 68:32; up to 25 mg/100 kcal of inositol; up to
9.0 mg/100 kcal of butyrate; and/or from about 1.8 .mu.g/100 kcal
to about 2.0 .mu.g/100 kcal of vitamin D; and C) providing a third
nutritional composition comprising: from about 0.6 mg/100 kcal to
about 1.2 mg/100 kcal of iron; up to about 2 mg/100 kcal of zinc;
up to about 3 g/100 kcal of protein; a whey:casein ratio of between
45:55 and 54:46; up to 20 mg/100 kcal of inositol; up to 9.0 mg/100
kcal of butyrate; and/or from about 1.8 .mu.g/100 kcal to about 2.0
.mu.g/100 kcal of vitamin D.
33. The staged feeding regimen of claim 32, wherein the first
nutritional composition comprises up to about 0.2 mg/100 kcal of
iron.
34. The staged feeding regimen of claim 32, wherein the first
nutritional composition comprises a whey:casein ratio of between
72:28 and 90:10.
35. A staged feeding regimen for an infant or child, comprising the
steps of: A) providing a first nutritional composition comprising
no greater than 9.0 mg/100 kcal of butyrate; and B) providing a
second nutritional composition comprising at least 9.0 mg/100 kcal
of butyrate.
36. The staged feeding regimen of claim 35, wherein the first
nutritional composition is provided to an infant from the age of
birth to three months and the second composition is provided to an
infant of age three months to twelve months.
37. The staged feeding regimen of claim 35, wherein the first
nutritional composition is provided to an infant from the age of
birth to six months and the second composition is provided to an
infant of age six months to twelve months.
38. The staged feeding regimen of claim 37 further comprising the
step of: C) providing a third nutritional composition comprising at
least 9.0 mg/100 kcal of butyrate, wherein the third nutritional
composition is provided to the infant after the age of twelve
months.
39. The staged feeding regimen of claim 35, wherein the first
nutritional composition and/or second nutritional composition
further comprise sialic acid, human milk oligosaccharides,
lactoferrin, or any combination thereof.
40. The staged feeding regimen of claim 39, wherein the level of
sialic acid, human milk oligosaccharides, and/or lactoferrin is
higher in the first nutritional composition than the second
nutritional composition.
Description
TECHNICAL FIELD
[0001] The present disclosure relates generally to nutritional
compositions comprising dietary butyrate and uses thereof. The
nutritional compositions are suitable for administration to
pediatric subjects. Further, disclosed are methods for reducing the
incidence of obesity and metabolic syndrome in target subjects.
Additionally, disclosed are methods for maintaining healthy weight
and metabolism in target subjects. Further disclosed are methods
for improving adipose tissue function and improving the quality of
adipose tissue in target subjects. The disclosed nutritional
compositions may provide additive and/or synergistic beneficial
health effects.
BACKGROUND ART
[0002] Obesity represents a major global health problem that is
frequently associated with chronic diseases such as type-2
diabetes. Furthermore, obesity is the hallmark of metabolic
syndrome. Childhood overweight and obesity has increased
substantially in the past two decades causing certain
cardiovascular, muscle/skeletal, and endocrine complications to
start in early childhood. Furthermore, there is an increase in the
incidence rates of non-alcoholic fatty liver disease and cognitive
impairment in children. Accordingly, there exists the need to
provide an infant formula or nutritional composition that is
capable of reducing the incidence of obesity and reducing the
incidence of symptoms and chronic diseases associated with
metabolic syndrome in infants and children.
[0003] Thus, provided herein are nutritional compositions
containing dietary butyrate in combination with other nutrients
that promote a healthy body weight and reduce the risk of obesity
when administered to a target subject, such as an infant. Further
provided are compositions for improving adipose tissue function and
improving the quality of adipose tissue in target subjects in
formula-fed infants.
BRIEF SUMMARY
[0004] Briefly, the present disclosure is directed, in an
embodiment, to a nutritional composition that includes dietary
butyrate. In some embodiments, the dietary butyrate may be provided
in the form of sodium butyrate, butyrate triglycerides,
encapsulated butyrate, or enriched lipid fractions from milk. In
some embodiments, the nutritional composition includes dietary
butyrate in combination with long chain polyunsaturated fatty
acids, such as docosahexaenoic acid and/or arachidonic acid, one or
more probiotics, such as Lactobacillus rhamnosus GG,
phosphatidylethanolamine (PE), sphingomyelin, inositol, vitamin D,
and combinations thereof.
[0005] The present disclosure further provides methods for
promoting and or maintaining a healthy weight in pediatric subjects
by administering the nutritional composition disclosed herein to
the pediatric subject.
[0006] It is to be understood that both the foregoing general
description and the following detailed description present
embodiments of the disclosure and are intended to provide an
overview or framework for understanding the nature and character of
the disclosure as it is claimed. The description serves to explain
the principles and operations of the claimed subject matter. Other
and further features and advantages of the present disclosure will
be readily apparent to those skilled in the art upon a reading of
the following disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 illustrates an example chromatogram of ethyl acetate
solvent blank.
[0008] FIG. 2 illustrates an example chromatogram of an internal
standard at 15 and butyric acid at 50 .mu.g/mL.
[0009] FIG. 3 illustrates an example chromatogram of NP H.sub.2O
blank through the sample preparation.
[0010] FIG. 4A illustrates a chromatogram of a human milk
sample
[0011] FIG. 4B illustrates the same chromatogram of a human milk
sample showing the internal standard and butyric acid (BA)
peaks.
[0012] FIG. 5 is a sample sequence setup template.
[0013] FIG. 6 illustrates a schematic process flow chart for the
procedure outlined in Example 4.
DETAILED DESCRIPTION
[0014] Reference now will be made in detail to the embodiments of
the present disclosure, one or more examples of which are set forth
herein below. Each example is provided by way of explanation of the
nutritional composition of the present disclosure and is not a
limitation. In fact, it will be apparent to those skilled in the
art that various modifications and variations can be made to the
teachings of the present disclosure without departing from the
scope of the disclosure. For instance, features illustrated or
described as part of one embodiment, can be used with another
embodiment to yield a still further embodiment.
[0015] Thus, it is intended that the present disclosure covers such
modifications and variations as come within the scope of the
appended claims and their equivalents. Other objects, features and
aspects of the present disclosure are disclosed in or are apparent
from the following detailed description. It is to be understood by
one of ordinary skill in the art that the present discussion is a
description of exemplary embodiments only and is not intended as
limiting the broader aspects of the present disclosure.
[0016] The present disclosure relates generally to nutritional
compositions comprising dietary butyrate in combination with other
nutrients disclosed herein. Additionally, the disclosure relates to
methods for maintaining healthy weight, reducing the incidence of
obesity, reducing the incidence of metabolic syndrome, improving
adipose tissue function, and improving the quality of adipose
tissue in target subjects.
[0017] "Nutritional composition" means a substance or formulation
that satisfies at least a portion of a subject's nutrient
requirements. The terms "nutritional (s)", "nutritional formula
(s)", "enteral nutritional (s)", and "nutritional supplement(s)"
are used as non-limiting examples of nutritional composition(s)
throughout the present disclosure. Moreover, "nutritional
composition(s)" may refer to liquids, powders, gels, pastes,
solids, tablets, capsules, concentrates, suspensions, or
ready-to-use forms of enteral formulas, oral formulas, formulas for
infants, formulas for pediatric subjects, formulas for children,
growing-up milks and/or formulas for adults.
[0018] "Pediatric subject" means a human less than 13 years of age.
In some embodiments, a pediatric subject refers to a human subject
that is between birth and 8 years old. In other embodiments, a
pediatric subject refers to a human subject between 1 and 6 years
of age. In still further embodiments, a pediatric subject refers to
a human subject between 6 and 12 years of age. The term "pediatric
subject" may refer to infants (preterm or fullterm) and/or
children, as described below.
[0019] "Infant" means a human subject ranging in age from birth to
not more than one year and includes infants from 0 to 12 months
corrected age. The phrase "corrected age" means an infant's
chronological age minus the amount of time that the infant was born
premature. Therefore, the corrected age is the age of the infant if
it had been carried to full term. The term infant includes low
birth weight infants, very low birth weight infants, and preterm
infants. "Preterm" means an infant born before the end of the 37th
week of gestation. "Full term" means an infant born after the end
of the 37th week of gestation.
[0020] "Child" means a subject ranging in age from 12 months to
about 13 years. In some embodiments, a child is a subject between
the ages of 1 and 12 years old. In other embodiments, the terms
"children" or "child" refer to subjects that are between one and
about six years old, or between about seven and about 12 years old.
In other embodiments, the terms "children" or "child" refer to any
range of ages between 12 months and about 13 years.
[0021] "Infant formula" means a composition that satisfies at least
a portion of the nutrient requirements of an infant. In the United
States, the content of an infant formula is dictated by the federal
regulations set forth at 21 C.F.R. Sections 100, 106, and 107.
[0022] The term "medical food" refers enteral compositions that are
formulated or intended for the dietary management of a disease or
disorder. A medical food may be a food for oral ingestion or tube
feeding (nasogastric tube), may be labeled for the dietary
management of a specific medical disorder, disease or condition for
which there are distinctive nutritional requirements, and may be
intended to be used under medical supervision.
[0023] The term "peptide" as used herein describes linear molecular
chains of amino acids, including single chain molecules or their
fragments. The peptides described herein include no more than 50
total amino acids. Peptides may further form oligomers or multimers
consisting of at least two identical or different molecules.
Furthermore, peptidomimetics of such peptides where amino acid (s)
and/or peptide bond (s) have been replaced by functional analogs
are also encompassed by the term "peptide". Such functional
analogues may include, but are not limited to, all known amino
acids other than the 20 gene-encoded amino acids such as
selenocysteine.
[0024] The term "peptide" may also refer to naturally modified
peptides where the modification is effected, for example, by
glycosylation, acetylation, phosphorylation and similar
modification which are well known in the art. In some embodiments,
the peptide component is distinguished from a protein source also
disclosed herein. Further, peptides may, for example, be produced
recombinantly, semi-synthetically, synthetically, or obtained from
natural sources such as after hydrolysation of proteins, including
but not limited to casein, all according to methods known in the
art.
[0025] The term "molar mass distribution" when used in reference to
a hydrolyzed protein or protein hydrolysate pertains to the molar
mass of each peptide present in the protein hydrolysate. For
example, a protein hydrolysate having a molar mass distribution of
greater than 500 Daltons means that each peptide included in the
protein hydrolysate has a molar mass of at least 500 Daltons.
Accordingly, in some embodiments, the peptides disclosed in Table 3
and Table 4 are derived from a protein hydrolysate having a molar
mass distribution of greater than 500 Daltons. To produce a protein
hydrolysate having a molar mass distribution of greater than 500
Daltons, a protein hydrolysate may be subjected to certain
filtering procedures or any other procedure known in the art for
removing peptides, amino acids, and/or other proteinaceous material
having a molar mass of less than 500 Daltons. For the purposes of
this disclosure, any method known in the art may be used to produce
the protein hydrolysate having a molar mass distribution of greater
than 500 Dalton.
[0026] The term "protein equivalent" or "protein equivalent source"
includes any protein source, such as soy, egg, whey, or casein, as
well as non-protein sources, such as peptides or amino acids.
Further, the protein equivalent source can be any used in the art,
e.g., nonfat milk, whey protein, casein, soy protein, hydrolyzed
protein, peptides, amino acids, and the like. Bovine milk protein
sources useful in practicing the present disclosure include, but
are not limited to, milk protein powders, milk protein
concentrates, milk protein isolates, nonfat milk solids, nonfat
milk, nonfat dry milk, whey protein, whey protein isolates, whey
protein concentrates, sweet whey, acid whey, casein, acid casein,
caseinate (e.g. sodium caseinate, sodium calcium caseinate, calcium
caseinate), soy bean proteins, and any combinations thereof. The
protein equivalent source can, in some embodiments comprise
hydrolyzed protein, including partially hydrolyzed protein and
extensively hydrolyzed protein. The protein equivalent source may,
in some embodiments, include intact protein. More particularly, the
protein source may include a) about 20% to about 80% of the peptide
component described herein, and b) about 20% to about 80% of an
intact protein, a hydrolyzed protein, or a combination thereof.
[0027] The term "protein equivalent source" also encompasses free
amino acids. In some embodiments, the amino acids may comprise, but
are not limited to, histidine, isoleucine, leucine, lysine,
methionine, cysteine, phenylalanine, tyrosine, threonine,
tryptophan, valine, alanine, arginine, asparagine, aspartic acid,
glutamic acid, glutamine, glycine, proline, serine, carnitine,
taurine and mixtures thereof. In some embodiments, the amino acids
may be branched chain amino acids. In certain other embodiments,
small amino acid peptides may be included as the protein component
of the nutritional composition. Such small amino acid peptides may
be naturally occurring or synthesized.
[0028] "Fractionation procedure" includes any process in which a
certain quantity of a mixture is divided up into a number of
smaller quantities known as fractions. The fractions may be
different in composition from both the mixture and other fractions.
Examples of fractionation procedures include but are not limited
to, melt fractionation, solvent fractionation, supercritical fluid
fractionation and/or combinations thereof.
[0029] "Milk fat globule membrane" includes components found in the
milk fat globule membrane including but not limited to milk fat
globule membrane proteins such as Mucin 1, Butyrophilin,
Adipophilin, CD36, CD14, Lactadherin (PAS6/7), Xanthine oxidase and
Fatty Acid binding proteins etc. Additionally, "milk fat globule
membrane" may include phospholipids, cerebrosides, gangliosides,
sphingomyelins, and/or cholesterol.
[0030] The term "growing-up milk" refers to a broad category of
nutritional compositions intended to be used as a part of a diverse
diet in order to support the normal growth and development of a
child between the ages of about 1 and about 6 years of age.
[0031] "Milk" means a component that has been drawn or extracted
from the mammary gland of a mammal. In some embodiments, the
nutritional composition comprises components of milk that are
derived from domesticated ungulates, ruminants or other mammals or
any combination thereof.
[0032] "Nutritionally complete" means a composition that may be
used as the sole source of nutrition, which would supply
essentially all of the required daily amounts of vitamins,
minerals, and/or trace elements in combination with proteins,
carbohydrates, and lipids. Indeed, "nutritionally complete"
describes a nutritional composition that provides adequate amounts
of carbohydrates, lipids, essential fatty acids, proteins,
essential amino acids, conditionally essential amino acids,
vitamins, minerals and energy required to support normal growth and
development of a subject.
[0033] A nutritional composition that is "nutritionally complete"
for a full term infant will, by definition, provide qualitatively
and quantitatively adequate amounts of all carbohydrates, lipids,
essential fatty acids, proteins, essential amino acids,
conditionally essential amino acids, vitamins, minerals, and energy
required for growth of the full term infant.
[0034] A nutritional composition that is "nutritionally complete"
for a child will, by definition, provide qualitatively and
quantitatively adequate amounts of all carbohydrates, lipids,
essential fatty acids, proteins, essential amino acids,
conditionally essential amino acids, vitamins, minerals, and energy
required for growth of a child.
[0035] "Exogenous butyrate" or "dietary butyrate" each refer to
butyrate or butyrate derivatives which are intentionally included
in the nutritional composition of the present disclosure itself,
rather than generated in the gut.
[0036] "Endogenous butyrate" or "butyrate from endogenous sources"
each refer to butyrate present in the gut as a result of ingestion
of the disclosed composition that is not added as such, but is
present as a result of other components or ingredients of the
composition; the presence of such other components or ingredients
of the composition stimulates butyrate production in the gut.
[0037] "Probiotic" means a microorganism with low or no
pathogenicity that exerts a beneficial effect on the health of the
host.
[0038] The term "non-viable probiotic" means a probiotic wherein
the metabolic activity or reproductive ability of the referenced
probiotic has been reduced or destroyed. More specifically,
"non-viable" or "non-viable probiotic" means non-living probiotic
microorganisms, their cellular components and/or metabolites
thereof. Such non-viable probiotics may have been heat-killed or
otherwise inactivated. The "non-viable probiotic" does, however,
still retain, at the cellular level, its cell structure or other
structure associated with the cell, for example exopolysaccharide
and at least a portion its biological glycol-protein and DNA/RNA
structure and thus retains the ability to favorably influence the
health of the host. Contrariwise, the term "viable" refers to live
microorganisms. As used herein, the term "non-viable" is synonymous
with "inactivated".
[0039] "Prebiotic" means a non-digestible food ingredient that
beneficially affects the host by selectively stimulating the growth
and/or activity of one or a limited number of bacteria in the
digestive tract that can improve the health of the host.
[0040] "Phospholipids" means an organic molecule that contains a
diglyceride, a phosphate group and a simple organic molecule.
Examples of phospholipids include but are not limited to,
phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, phsphatidylinositol, phosphatidylinositol
phosphate, phosphatidylinositol biphosphate and
phosphatidylinositol triphosphate, ceramide phosphorylcholine,
ceramide phosphorylethanolamine and ceramide phosphoryl glycerol.
This definition further includes sphingolipids such as
sphingomyelin. Glycosphingolipds are quantitatively minor
constituents of the MFGM, and consist of cerebrosides (neutral
glycosphingolipids containing uncharged sugars) and gangliosides.
Gangliosides are acidic glycosphingolipids that contain sialic acid
(N-acetylneuraminic acid (NANA)) as part of their carbohydrate
moiety. There are various types of gangliosides originating from
different synthetic pathways, including GM3, GM2, GM1a, GD1a, GD3,
GD2, GD1b, Glib and GQ1b (Fujiwara et al., 2012). The principal
gangliosides in milk are GM3 and GD3 (Pan & Izumi, 1999). The
different types of gangliosides vary in the nature and length of
their carbohydrate side chains, and the number of sialic acid
attached to the molecule.
[0041] "Alpha-lipoic acid", abbreviated "ALA" herein, refers to an
organosulfur compound derived from octanoic acid having the
molecular formula C.sub.8H.sub.14S.sub.2O.sub.2. Generally, ALA
contains two sulfur atoms attached via a disulfide bond.
Alpha-lipoic acid is synonymous with lipoic acid, abbreviated "LA",
and the two terms and abbreviations may be used interchangeable
herein.
[0042] As used herein "sulforaphane" includes any known isomers of
sulforaphane including but not limited to L-sulforaphane. In some
embodiments, sulforaphane may include only L-sulforaphane while, in
other embodiments, the reference to sulforaphane may include
L-sulforaphane, D-sulforaphane, any other suitable isomer of
sulforaphane, and any combinations thereof. Accordingly, the term
sulforaphane as used herein includes any isomers of sulforaphane
including, but not limited to, stereoisomers, optical isomers,
structural isomers, enantiomers, geometric isomers, and
combinations thereof.
[0043] The nutritional composition of the present disclosure may be
substantially free of any optional or selected ingredients
described herein, provided that the remaining nutritional
composition still contains all of the required ingredients or
features described herein. In this context, and unless otherwise
specified, the term "substantially free" means that the selected
composition may contain less than a functional amount of the
optional ingredient, typically less than 0.1% by weight, and also,
including zero percent by weight of such optional or selected
ingredient.
[0044] All percentages, parts and ratios as used herein are by
weight of the total composition, unless otherwise specified.
[0045] All references to singular characteristics or limitations of
the present disclosure shall include the corresponding plural
characteristic or limitation, and vice versa, unless otherwise
specified or clearly implied to the contrary by the context in
which the reference is made.
[0046] All combinations of method or process steps as used herein
can be performed in any order, unless otherwise specified or
clearly implied to the contrary by the context in which the
referenced combination is made.
[0047] The methods and compositions of the present disclosure,
including components thereof, can comprise, consist of, or consist
essentially of the essential elements and limitations of the
embodiments described herein, as well as any additional or optional
ingredients, components or limitations described herein or
otherwise useful in nutritional compositions.
[0048] As used herein, the term "about" should be construed to
refer to both of the numbers specified as the endpoint(s) of any
range. Any reference to a range should be considered as providing
support for any subset within that range.
[0049] The present disclosure is directed to nutritional
compositions including dietary butyrate. Non-limiting examples of
butyrate for use herein include butyric acid, butyrate salts,
glycerol esters of butyric acid, and amide derivatives of amino
acids. The nutritional compositions may further include a
carbohydrate source, a protein source, and a fat or lipid source.
In some embodiments, the nutritional compositions may include a
component capable of stimulating endogenous butyrate production; in
other embodiments, the nutritional compositions may include both
dietary and endogenous butyrate.
[0050] The benefit to providing dietary butyrate in combination
with selected nutrients herein is healthy weight development and
metabolism, in particular improving adipose tissue function and
quality. Furthermore, providing dietary butyrate in combination
with selected nutrients may provide anti-inflammatory properties,
such as a reduction in the inflammatory processes in fat tissues,
the liver, and the brain.
[0051] Indeed, dietary butyrate may affect energy homeostasis,
glucose metabolism, and insulin sensitivity. Dietary
supplementation with dietary butyrate may prevent the developments
of diet-induced insulin resistance and improve insulin sensitivity,
thus promoting healthy metabolic programming and reducing the risk
of metabolic syndrome. Further, providing dietary butyrate may
reduce insulin resistance and reduce obesity-associated
inflammation. Without being bound by any particular theory,
mechanistically dietary butyrate acts through promotion of
mitochondrial energy expenditure and modulation of the inflammatory
response. These mechanisms may be involved in maintaining healthy
weight during infancy and pediatric development.
[0052] In certain embodiments, the dietary butyrate is incorporated
into a nutritional composition that is an infant formula.
Currently, many infant formulas are not formulated with dietary
butyrate or are not formulated with effective amounts of dietary
butyrate for providing a beneficial health effect once administered
to the infant. One reason that infant formulas include little to no
dietary butyrate is due to the unpleasant organoleptic properties
exhibited by the nutritional composition when butyrate compounds
are incorporated into the nutritional composition. For example,
many butyrate compounds exhibit an odor that makes consuming the
nutritional composition in which they are incorporated an
unpleasant experience. Furthermore, the pediatric and infant
population will not readily consume nutritional products having an
unpleasant odor, taste, and/or mouthfeel. Accordingly, there exists
a need for a nutritional composition formulated for administration
to a pediatric subject or an infant that provides butyrate yet does
not have diminished organoleptic properties. The incorporation of
the dietary butyrate compounds disclosed herein into pediatric and
infant nutritional compositions will provide butyrate while still
providing a pleasant sensory experience.
[0053] Accordingly, given that dietary butyrate is not supplemented
in effective levels in infant formula, many formula-fed infants may
not obtain enough butyrate through diet in comparison to breast-fed
infants. Accordingly, providing the dietary butyrate in an infant
formula and administering the formula to a pediatric subject
ensures that certain risk factors for cardiovascular disease and
metabolic syndrome may be further reduced in formula-fed infants as
compared to breast-fed infants. Furthermore, providing the
compositions herein to an infant or child may strengthen the
capacity and flexibility of fat tissues.
[0054] In some embodiments, the nutritional composition includes a
source of dietary butyrate that is present in an amount of from
about 0.01 mg/100 Kcal to about 300 mg/100 Kcal. In some
embodiments, the nutritional composition includes a source of
dietary butyrate that is present in an amount of from about 0.1
mg/100 Kcal to about 300 mg/100 Kcal. In some embodiments, the
nutritional composition includes a source of dietary butyrate that
is present in an amount of from about 0.1 mg/100 Kcal to about 300
mg/100 Kcal. In some embodiments, the nutritional composition
includes a source of dietary butyrate that is present in an amount
of from about 1 mg/100 Kcal to about 275 mg/100 Kcal. In some
embodiments, the nutritional composition includes a source of
dietary butyrate that is present in an amount of from about 5
mg/100 Kcal to about 200 mg/100 Kcal. In some embodiments, the
nutritional composition includes a source of dietary butyrate that
is present in an amount of from about 10 mg/100 Kcal to about 150
mg/100 Kcal. In some embodiments the amount of butyrate is from
about 0.6 mg/100 kcal to about 6.1 mg per 100 kcal.
[0055] In some embodiments, the nutritional composition includes a
source of dietary butyrate that is present in an amount based on
the weight percentage of total fat. Accordingly, in some
embodiments the nutritional composition includes from about 0.2 mg
to about 57 mg of dietary butyrate per gram of fat in the
nutritional composition. In some embodiments, the nutritional
compositions includes from about 1 mg to about 50 mg of dietary
butyrate per gram of fat in the nutritional composition. Still, in
some embodiments the nutritional composition includes from about 5
mg to about 40 mg of dietary butyrate per gram of fat in the
nutritional composition. In certain embodiments, the nutritional
composition includes from about 10 mg to about 30 mg of dietary
butyrate per gram of fat in the nutritional composition.
[0056] In some embodiments, the nutritional composition includes a
source of dietary butyrate that is present in an amount based on a
liter of formula. In some embodiments, the nutritional composition
includes from about 0.6 mg to about 2100 mg of dietary butyrate per
Liter of nutritional composition. In some embodiments, the
nutritional composition includes from about 2 mg to about 2000 mg
of dietary butyrate per Liter of nutritional composition. In some
embodiments, the nutritional composition includes from about 10 mg
to about 1800 mg of dietary butyrate per Liter of nutritional
composition. In some embodiments, the nutritional composition
includes from about 25 mg to about 1600 mg of dietary butyrate per
Liter of nutritional composition. In some embodiments, the
nutritional composition includes from about 40 mg to about 1400 mg
of dietary butyrate per Liter of nutritional composition. In some
embodiments, the nutritional composition includes from about 50 mg
to about 1200 mg of dietary butyrate per Liter of nutritional
composition. In some embodiments, the nutritional composition
includes from about 100 mg to about 1000 mg of dietary butyrate per
Liter of nutritional composition.
[0057] In some embodiments the dietary butyrate is provided by one
or more of the following: butyric acid; butyrate salts, including
sodium butyrate, potassium butyrate, calcium butyrate, and/or
magnesium butyrate; glycerol esters of butyric acid; and/or amide
derivative of butyric acid.
[0058] The dietary butyrate can be supplied by any suitable source
known in the art. Non-limiting sources of dietary butyrate includes
animal source fats and derived products, such as but not limited to
milk, milk fat, butter, buttermilk, butter serum, cream; microbial
fermentation derived products, such as but not limited to yogurt
and fermented buttermilk; and plant source derived seed oil
products, such as pineapple and/or pineapple oil, apricot and/or
apricot oil, barley, oats, brown rice, bran, green beans, legumes,
leafy greens, apples, kiwi, oranges. In some embodiments, the
dietary butyrate is synthetically produced. In embodiments where
the dietary butyrate is synthetically produced, the chemical
structure of the dietary butyrate may be modified as necessary.
Further, the dietary butyrate produced synthetically can be
purified by any means known in the art to produce a purified
dietary butyrate additive that can be incorporated into the
nutritional compositions disclosed herein. The dietary butyrate may
be provided by dairy lipids and/or triglyceride bound forms of
butyrate.
[0059] In some embodiments, the dietary butyrate may be provided in
an encapsulated form. In certain embodiments, the encapsulation of
the dietary butyrate may provide for longer shelf-stability and may
provide for improved organoleptic properties of the nutritional
composition. For example, in some embodiments, the dietary butyrate
may be encapsulated or coated by the use of, or combination of, fat
derived materials, such as mono- and di-glycerides; sugar and acid
esters of glycerides; phospholipids; plant, animal and microbial
derived proteins and hydrocolloids, such as starches,
maltodextrins, gelatin, pectins, glucans, caseins, soy proteins,
and/or whey proteins.
[0060] The dietary butyric acid may also be provided in a coated
form. For example, coating certain glycerol esters of butyric acids
with fat derived materials, such as mono- and di-glycerides; sugar
and acid esters of glycerides; phospholipids; plant, animal and
microbial derived proteins and hydrocolloids, such as starches,
maltodextrins, gelatin, pectins, glucans, caseins, soy proteins,
and/or whey proteins may improve the shelf-stability of the dietary
butyrate and may further improve the overall organoleptic
properties of the nutritional composition.
[0061] In certain embodiments, the dietary butyrate comprises
alkyl, and or glycerol esters of butyric acid. Glycerol esters of
butyric acid may offer minimal complexity when formulated and
processed in the nutritional composition. Additionally, glycerol
esters of butyric acid may improve the shelf life of the
nutritional composition including dietary butyrate an may further
have a low impact on the sensory attributes of the finished
product.
[0062] The dietary butyrate comprises amide derivatives of butyric
acid in some embodiments. Generally, these amide derivatives of
butyric acid are a solid, odorless, and tasteless form and are more
stable than certain butyric acid esters at gastric pH. Further, the
amide derivatives of butyric acid are able to release the
corresponding acid by alkaline hydrolysis in the small and large
intestine, thereby allowing for absorption of the dietary
butyrate.
[0063] In some embodiments, the dietary butyrate may comprise
butyrate salts, for example, sodium butyrate, potassium butyrate,
calcium butyrate, magnesium butyrate, and combinations thereof. In
some embodiments, the use of selected dietary butyrate salts may
improve intestinal health when provided to target subjects. In
certain embodiments, dietary butyrate comprises a suitable butyrate
salt that has been coated with one or more fats or lipids. In
certain embodiments wherein the dietary butyrate comprises a fat
coated butyrate salt, the nutritional composition may be a
dry-powdered composition into which the dietary butyrate is
incorporated.
[0064] In some embodiments, the dietary butyrate may comprise any
of the butyrate compounds disclosed herein that are formulated to
be in complex form with chitosan or one or cyclodextrins. For
example, cyclodextrins are cyclic oligosaccharides composed of six
(a-cyclodextrin), seven (.beta.-cyclodextrin), or eight
(gamma-cyclodextrin) units of a-1,4-glucopyranose. Cyclodextrins
are further characterized by a hydrophilic exterior surface and a
hydrophobic core. Without being bound by any particular theory, the
aliphatic butyrate chain would form a complex with the cyclodextrin
core, thus increasing its molecular weight and, thus, reducing the
volatility of the butyrate compound. Accordingly, the
bioavailability of dietary butyrate may be improved when the
dietary butyrate includes butyrate compounds in complex form with
one or more cyclodextrins. Further, cyclodextrins are bulky
hydrophobic molecules that are resistant to stomach acid as well as
gastrointestinal enzymes, thus administration of the
butyrate-cyclodextrin complex as described herein would promote
absorption of the dietary butyrate in the small intestines.
[0065] In some embodiments the dietary butyrate is provided from an
enriched lipid fraction derived from milk. For example, bovine milk
fat has a butyric acid content that may be 20 times higher than the
butyric acid content in human milk fat. Furthermore, among the
short chain fatty acids ("SCFAs") present in human milk, i.e. fatty
acids having a carbon chain length from 4 to 12, butyric acid (C4)
is one of the most predominant in bovine milk. As such, bovine milk
fat and/or enriched fractions of bovine milk fat may be included in
a nutritional composition to provide dietary butyrate.
[0066] In embodiments where the dietary butyrate is provided by an
enriched lipid fraction derived from milk the enriched lipid
fraction derived from milk may be produced by any number of
fractionation techniques. These techniques include but are not
limited to melting point fractionation, organic solvent
fractionation, super critical fluid fractionation, and any variants
and combinations thereof.
[0067] Furthermore, mixtures that may be subjected to the
fractionation procedures to produce the enriched lipid fraction
include, but are not limited to, bovine whole milk, bovine cream,
caprine milk, ovine milk, yak milk, and/or mixtures thereof. In a
preferred embodiment the milk mixture used to create the enriched
lipid fraction is bovine milk.
[0068] In addition to providing dietary butyrate, the enriched
lipid fraction may comprise an one of the following ingredients:
saturated fatty acids; trans-fatty acids; branched-chain fatty
acids ("BCFAs"), including odd-branched chain fatty acids
("OBCFAs"); conjugated linoleic acid ("CLA"); monounsaturated fatty
acids; polyunsaturated fatty acids; cholesterol; phospholipids; and
milk fat globule membrane, including milk fat globule membrane
protein.
[0069] In some embodiments the enriched lipid fraction includes,
per 100 Kcal, one or more of the following:
[0070] from about 0.1 g to 8.0 g of saturated fatty acids;
[0071] from about 0.2 g to 7.0 g trans-fatty acids;
[0072] from about 0.003 g to about 6.1 g branched-chain fatty
acids;
[0073] from about 0.026 g to about 2.5 g conjugated linoleic
acid;
[0074] from about 0.8 g to about 2.5 g monounsaturated fatty
acids;
[0075] from about 2.3 g to about 4.4 g polyunsaturated fatty
acids;
[0076] from about 100 mg to about 400 mg of cholesterol;
[0077] from about 50 mg to about 400 mg of phospholipids; and/or
from about 10 mg to about 500 mg of milk fat globule membrane.
[0078] The following example illustrates a milk fat fraction having
an enriched concentration of butyric acid (C4) that may be produced
by a fractionation procedure.
Example 1
[0079] Illustrated in Table 1 below is a lipid profile of
fractionated milk fat produced by super critical carbon extraction
fractionation procedure and by melt-fractionation.
TABLE-US-00001 TABLE 1 Milk Fat composition (g fatty acid /100 g
TOTAL fatty acids) AMF SCCO 2 MeltFrac 10 C. C 4:0 3.9 6.0 4.7 C
6:0 2.5 3.3 2.9 C 8:0 1.4 1.9 1.8 C 10:0 3.1 3.9 3.8 C 12:0 4.2 4.1
4.8 C 14:0 11.4 12.2 10.9 C 14:1 1.1 1.0 1.3 C 15:0 1.1 1.0 0.9 C
16:0 29.4 29.6 22.3 C 16:1 1.9 1.4 2.2 C 17:0 0.6 0.5 0.4 C 18:0
11.4 8.2 6.1 C 18:1, cis, .omega.9 21.9 16.5 25.3 C 18:1, trans,
.omega.9 0.3 1.6 1.9 C 18:2, .omega. 6 1.9 2.2 1.9 C 18:3, .omega.
3, a 0.6 0.4 0.6 C 20:0 0.0 0.1 0.1 C 20:1, .omega.9 0.1 0.1 0.2
Saturated 68.7 70.7 58.6 Unsaturated 27.8 23.1 33.3 AMF = anhydrous
milk fat; SCCO2 = super-critical carbon dioxide fraction (super
olein). MeltFrac = melt crystallization fraction separated at
10.degree. C.
[0080] In some embodiments, the nutritional composition may include
an enriched milk product, such as an enriched whey protein
concentrate (eWPC). Enriched milk product generally refers to a
milk product that has been enriched with certain milk fat globule
membrane (MFGM) components, such as proteins and lipids found in
the MFGM. The enriched milk product can be formed by, e.g.,
fractionation of non-human (e.g., bovine) milk. Enriched milk
products have a total protein level which can range between 20% and
90%, more preferably between 68% and 80%, of which between 3% and
50% is MFGM proteins; in some embodiments, MFGM proteins make up
from 7% to 13% of the enriched milk product protein content.
Enriched milk products also comprise from 0.5% to 5% (and, at
times, 1.2% to 2.8%) sialic acid, from 2% to 25% (and, in some
embodiments, 4% to 10%) phospholipids, from 0.4% to 3%
sphingomyelin, from 0.05% to 1.8%, and, in certain embodiments
0.10% to 0.3%, gangliosides and from 0.02% to about 1.2%, more
preferably from 0.2% to 0.9%, cholesterol. Thus, enriched milk
products include desirable components at levels higher than found
in bovine and other non-human milks.
[0081] In some embodiments, the enriched milk product may contain
certain polar lipids such as (1) Glycerophospholipids such as
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylserine (PS), and phosphatidylinositol (PI), and their
derivatives and (2) Sphingoids or sphingolipids such as
sphingomyelin (SM) and glycosphingolipids comprising cerebrosides
(neutral glycosphingolipids containing uncharged sugars) and the
gangliosides (GG, acidic glycosphingolipids containing sialic acid)
and their derivatives.
[0082] PE is a phospholipid found in biological membranes,
particularly in nervous tissue such as the white matter of brain,
nerves, neural tissue, and in spinal cord, where it makes up 45% of
all phospholipids. Sphingomyelin is a type of sphingolipid found in
animal cell membranes, especially in the membranous myelin sheath
that surrounds some nerve cell axons. It usually consists of
phosphocholine and ceramide, or a phosphoethanolamine head group;
therefore, sphingomyelins can also be classified as
sphingophospholipids. In humans, SM represents .about.85% of all
sphingolipids, and typically makes up 10-20 mol % of plasma
membrane lipids. Sphingomyelins are present in the plasma membranes
of animal cells and are especially prominent in myelin, a
membranous sheath that surrounds and insulates the axons of some
neurons.
[0083] In some embodiments, the enriched milk product includes
eWPC. The eWPC may be produced by any number of fractionation
techniques. These techniques include but are not limited to melting
point fractionation, organic solvent fractionation, super critical
fluid fractionation, and any variants and combinations thereof.
Alternatively, eWPC is available commercially, including under the
trade names Lacprodan MFGM-10 and Lacprodan PL-20, both available
from Arla Food Ingredients of Viby, Denmark. With the addition of
eWPC, the lipid composition of infant formulas and other pediatric
nutritional compositions can more closely resemble that of human
milk. For instance, the theoretical values of phospholipids (mg/L)
and gangliosides (mg/L) in an exemplary infant formula which
includes Lacprodan MFGM-10 or Lacprodan PL-20 can be calculated as
shown in Table 2:
TABLE-US-00002 TABLE 2 Total Item milk PL SM PE PC PI PS Other PL
GD3 MFGM-10 330 79.2 83.6 83.6 22 39.6 22 10.1 PL-20 304 79 64 82
33 33 12.2 8.5 PL: phospholipids; SM: sphingomyelin; PE:
phosphatidyl ethanolamine; PC: phosphatidyl choline; PI:
phosphatidyl inositol; PS: phosphatidyl serine; GD3: ganglioside
GD3.
[0084] In some embodiments, the eWPC is included in the nutritional
composition of the present disclosure at a level of about 0.5 grams
per liter (g/L) to about 10 g/L; in other embodiments, the eWPC is
present at a level of about 1 g/L to about 9 g/L. In still other
embodiments, eWPC is present in the nutritional composition at a
level of about 3 g/L to about 8 g/L. Alternatively, in certain
embodiments, the eWPC is included in the nutritional composition of
the present disclosure at a level of about 0.06 grams per 100 Kcal
(g/100 Kcal) to about 1.5 g/100 Kcal; in other embodiments, the
eWPC is present at a level of about 0.3 g/100 Kcal to about 1.4
g/100 Kcal. In still other embodiments, the eWPC is present in the
nutritional composition at a level of about 0.4 g/100 Kcal to about
1 g/100 Kcal.
[0085] Total phospholipids in the nutritional composition disclosed
herein (i.e., including phospholipids from the eWPC as well as
other components, but not including phospholipids from plant
sources such as soy lecithin, if used) is in a range of about 50
mg/L to about 2000 mg/L; in some embodiments it is about 100 mg/L
to about 1000 mg/L, or about 150 mg/L to about 550 mg/L. In certain
embodiments, the eWPC component also contributes sphingomyelin in a
range of about 10 mg/L to about 200 mg/L; in other embodiments, it
is about 30 mg/L to about 150 mg/L, or about 50 mg/L to about 140
mg/L. And, the eWPC can also contribute gangliosides, which in some
embodiments, are present in a range of about 2 mg/L to about 40
mg/L, or, in other embodiments about 6 mg/L to about 35 mg/L. In
still other embodiments, the gangliosides are present in a range of
about 9 mg/L to about 30 mg/L. In some embodiments, total
phospholipids in the nutritional composition (again not including
phospholipids from plant sources such as soy lecithin) is in a
range of about 6 mg/100 Kcal to about 300 mg/100 Kcal; in some
embodiments it is about 12 mg/100 Kcal to about 150 mg/100 Kcal, or
about 18 mg/100 Kcal to about 85 mg/100 Kcal. In certain
embodiments, the eWPC also contributes sphingomyelin in a range of
about 1 mg/100 Kcal to about 30 mg/100 Kcal; in other embodiments,
it is about 3.5 mg/100 Kcal to about 24 mg/100 Kcal, or about 6
mg/100 Kcal to about 21 mg/100 Kcal. And, gangliosides can be
present in a range of about 0.25 mg/100 Kcal to about 6 mg/100
Kcal, or, in other embodiments about 0.7 mg/100 Kcal to about 5.2
mg/100 Kcal. In still other embodiments, the gangliosides are
present in a range of about 1.1 mg/100 Kcal to about 4.5 mg/100
Kcal.
[0086] In some embodiments, the eWPC contains sialic acid (SA).
Generally, the term sialic acid (SA) is used to generally refer to
a family of derivatives of neuraminic acid. N-acetylneuraminic acid
(Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are among the most
abundant naturally found forms of SA, especially Neu5Ac in human
and cow's milk. Mammalian brain tissue contains the highest levels
of SA because of its incorporation into brain-specific proteins
such as neural cell adhesion molecule (NCAM) and lipids (e.g.,
gangliosides). It is considered that SA plays a role in neural
development and function, learning, cognition, and memory
throughout the life. In human milk, SA exists as free and bound
forms with oligosaccharides, protein and lipid. The content of SA
in human milk varies with lactation stage, with the highest level
found in colostrum. However, most SA in bovine milk is bound with
proteins, compared to the majority of SA in human milk bound to
free oligosaccharides. Sialic acid can be incorporated in to the
disclosed nutritional composition as is, or it can be provided by
incorporating casein glycomacropeptide (cGMP) having enhanced
sialic acid content, as discussed in U.S. Pat. Nos. 7,867,541 and
7,951,410, the disclosure of each of which are incorporated by
reference herein.
[0087] When present, sialic acid can be incorporated into the
nutritional composition of the present disclosure at a level of
about 100 mg/L to about 800 mg/L, including both inherent sialic
acid from the eWPC and exogenous sialic acid and sialic acid from
sources such as cGMP. In some embodiments, sialic acid is present
at a level of about 120 mg/L to about 600 mg/L; in other
embodiments the level is about 140 mg/L to about 500 mg/L. In
certain embodiments, sialic acid may be present in an amount from
about 1 mg/100 Kcals to about 120 mg/100 Kcal. In other embodiments
sialic acid may be present in an amount from about 14 mg/100 Kcal
to about 90 mg/100 Kcal. In yet other embodiments, sialic acid may
be present in an amount from about 15 mg/100 Kcal to about 75
mg/100 Kcal.
[0088] In certain embodiments, the nutritional composition may
further include inositol. Without being bound by any particular
theory, it has been found that nutritional supplementation of
inositol represents a feasible and effective approach to promote
oligodendrocyte survival and proliferation in a dose dependent
manner, resulting in a consistent increase in the number of
oligodendrocyte precursor cells. Accordingly, providing a
nutritional composition having a combination of dietary butyrate
and inositol may act synergistically to promote oligodendrocyte
survival and proliferation of OPCs into oligodendric cells.
Accordingly, nutritional supplementation with inositol provides
benefits for enhanced developmental myelination by which it
translates into a fundamental benefit for brain development. Given
the importance of functional myelination, nutritional
supplementation of inositol in combination with dietary butyrate is
beneficial to pediatric subjects by enhancing brain development and
health.
[0089] Furthermore, it is noted that the inclusion of dietary
butyrate into nutritional compositions, such as pediatric
compositions and infant formulas, may provide undesirable sensory
characteristics, such as poor taste and smell. Indeed, dietary
butyrate is generally not supplemented in effective levels given
the negative organoleptic properties that result. However, the
combination of inositol with dietary butyrate provide a nutritional
composition with improved organoleptic properties, such as improved
taste, because the sweet taste of inositol provides further
advantages in terms of palatability to pediatric consumers. Thus,
incorporating the combination of dietary and inositol into the
nutritional composition provides a nutritional composition with
improved organoleptic properties.
[0090] As such, in certain embodiments, inositol is present in the
nutritional compositions of the present disclosure at a level of at
least about 4 mg/100 Kcal; in other embodiments, inositol should be
present at a level of no greater than about 70 mg/100 Kcal. In
still other embodiments, the nutritional composition comprises
inositol at a level of about 5 mg/100 Kcal to about 65 mg/100 Kcal.
In a further embodiment, inositol is present in the nutritional
composition at a level of about 7 mg/100 Kcal to about 50 mg/100
Kcal. Moreover, inositol can be present as exogenous inositol or
inherent inositol. In embodiments, a major fraction of the inositol
(i.e., at least 40%) is exogenous inositol. In certain embodiments,
the ratio of exogenous to inherent inositol is at least 50:50; in
other embodiments, the ratio of exogenous to inherent inositol is
at least 60:40.
[0091] In certain embodiments, the nutritional composition may
further include at least one organosulfur compound including,
alpha-lipoic acid (ALA), allyl sulfide, allyl disulfide,
sulforaphane (SFN), L-sulforaphane (L-SFN), and combinations
thereof.
[0092] Allyl sulfide, also commonly known as diallyl sulfide is an
organosulfur compound with the chemical formula C.sub.6H.sub.10S.
Allyl sulfides, for example diallyl sulfide, diallyl disulfide, and
diallyl trisulfide, are principle constituents of garlic oil. In
vivo allyl sulfide may be converted to diallyl sulfoxide and
diallyl sulfone by cytochrome P450 2E1 (CYP2E1).
[0093] Sulforaphane (SFN) is a molecule within the isothiocyanate
group of organosulfur compounds having the molecular formula
C.sub.6H.sub.11NOS.sub.2. SFN and its isomers, for example
L-Sulforaphane ("L-SFN"), are known to exhibit anti-cancer and
antimicrobial properties in experimental models. SFN may be
obtained from cruciferous vegetables, such as broccoli, Brussels
sprouts or cabbage. SFN is produced when the enzyme myrosinase
reacts with glucoraphanin, a glucosinolate, transforming
glucoraphanin into SFN.
[0094] In some embodiments, the at least one organosulfur compound
incorporated into the nutritional composition comprises ALA.
Examples of ALA suitable for use in the nutritional composition
disclosed herein include, but are not limited to, enantiomers and
racemic mixtures of ALA, including, R-lipoic acid "RLA", S-lipoic
acid "SLA", and R/S-LA. Also suitable is R-lipoic acid stabilized
with either sodium ("Na-RALA") or potassium as
Potassium-R-Lipoate.
[0095] When incorporated into a nutritional composition for
practicing the method of the present disclosure, ALA may be present
in the nutritional composition, in some embodiments in an amount
from about 0.1 mg/100 Kcals to about 35 mg/100 Kcals. In some
embodiments, ALA may be present in an amount from about 2.0 mg/100
Kcals to about 25 mg/100 Kcals. In still other embodiments, ALA may
be present in an amount from about 5.0 mg/100 Kcals to about 15
mg/100 Kcals.
[0096] In some embodiments, the organosulfur compound incorporated
into the nutritional composition is allyl disulfide. Allyl
disulfide may be present in the nutritional composition, in some
embodiments, in an amount from about 1 mg/100 Kcals to about 170
mg/100 Kcals. In still some embodiments, allyl disulfide may be
present from about 50 mg/100 Kcals to about 120 mg/100 Kcals. In
still other embodiments, allyl disulfide may be present from about
75 mg/100 Kcals to about 100 mg/100 Kcals.
[0097] Sulforaphane, which includes L-sulforaphane, may be
incorporated into the nutritional composition in an amount from
about 1.5 mg/100 Kcals to about 7.5 mg/100 Kcals. Still in some
embodiments, sulforaphane may be present in an amount from about 2
mg/100 Kcals to about 6 mg/100 Kcals. In some embodiments,
sulforaphane may be present in an amount from about 3 mg/100 Kcals
to about 5 mg/100 Kcals.
[0098] In some embodiments, the nutritional composition comprises a
source of flavan-3-ols. Flavan-3-ols which are suitable for use in
the inventive nutritional composition include catechin, epicatechin
(EC), gallocatechin, epigallocatechin (EGC), epicatechin gallate
(ECG), epicatechin-3-gallate, epigallocatechin gallate (EGCG), and
combinations thereof. In certain embodiments, the nutritional
composition comprises EGCG.
[0099] In some embodiments, EGCG may be present in the nutritional
composition in an amount from about 0.01 mg/100 Kcal to about 18
mg/100 Kcal. In some embodiments, EGCG may be present in an amount
of from about 0.06 mg/100 Kcal to about 10 mg/100 Kcal. In some
embodiments, EGCG may be present in an amount of from about 0.10
mg/100 Kcal to about 5.0 mg/100 Kcal. In some embodiments, EGCG may
be present in an amount of from about 0.90 mg/100 Kcal to about 3.0
mg/100 Kcal.
[0100] The nutritional composition of the present disclosure also
includes at least one probiotic; in a preferred embodiment, the
probiotic comprises Lactobacillus rhamnosus GG ("LGG") (ATCC
53103). In certain other embodiments, the probiotic may be selected
from any other Lactobacillus species, Bifidobacterium species,
Bifidobacterium longum BB536 (BL999, ATCC: BAA-999),
Bifidobacterium longum AH1206 (NCIMB: 41382), Bifidobacterium breve
AH1205 (NCIMB: 41387), Bifidobacterium infantis 35624 (NCIMB:
41003), and Bifidobacterium animalis subsp. lactis BB-12 (DSM No.
10140) or any combination thereof.
[0101] The amount of the probiotic may vary from about
1.times.10.sup.4 to about 1.5.times.10.sup.12 cfu of probiotic(s)
per 100 Kcal. In some embodiments the amount of probiotic may be
from about 1.times.10.sup.6 to about 1.times.10.sup.9 cfu of
probiotic(s) per 100 Kcal. In certain other embodiments the amount
of probiotic may vary from about 1.times.10.sup.7 cfu/100 Kcal to
about 1.times.10.sup.8 cfu of probiotic(s) per 100 Kcal.
[0102] As noted, in a preferred embodiment, the probiotic comprises
LGG. LGG is a probiotic strain isolated from healthy human
intestinal flora. It was disclosed in U.S. Pat. No. 5,032,399 to
Gorbach, et al., which is herein incorporated in its entirety, by
reference thereto. LGG is resistant to most antibiotics, stable in
the presence of acid and bile, and attaches avidly to mucosal cells
of the human intestinal tract. It survives for 1-3 days in most
individuals and up to 7 days in 30% of subjects. In addition to its
colonization ability, LGG also beneficially affects mucosal immune
responses. LGG is deposited with the depository authority American
Type Culture Collection ("ATCC") under accession number ATCC
53103.
[0103] In an embodiment, the probiotic(s) may be viable or
non-viable. The probiotics useful in the present disclosure may be
naturally-occurring, synthetic or developed through the genetic
manipulation of organisms, whether such source is now known or
later developed.
[0104] In some embodiments, the nutritional composition may include
a source comprising probiotic cell equivalents, which refers to the
level of non-viable, non-replicating probiotics equivalent to an
equal number of viable cells. The term "non-replicating" is to be
understood as the amount of non-replicating microorganisms obtained
from the same amount of replicating bacteria (cfu/g), including
inactivated probiotics, fragments of DNA, cell wall or cytoplasmic
compounds. In other words, the quantity of non-living,
non-replicating organisms is expressed in terms of cfu as if all
the microorganisms were alive, regardless whether they are dead,
non-replicating, inactivated, fragmented etc. In non-viable
probiotics are included in the nutritional composition, the amount
of the probiotic cell equivalents may vary from about
1.times.10.sup.4 to about 1.5.times.10.sup.10 cell equivalents of
probiotic(s) per 100 Kcal. In some embodiments the amount of
probiotic cell equivalents may be from about 1.times.10.sup.6 to
about 1.times.10.sup.9 cell equivalents of probiotic(s) per 100
Kcal nutritional composition. In certain other embodiments the
amount of probiotic cell equivalents may vary from about
1.times.10.sup.7 to about 1.times.10.sup.8 cell equivalents of
probiotic(s) per 100 Kcal of nutritional composition.
[0105] In some embodiments, the probiotic source incorporated into
the nutritional composition may comprise both viable colony-forming
units, and non-viable cell-equivalents.
[0106] While, probiotics may be helpful in pediatric patients, the
administration of viable bacteria to pediatric subjects, and
particularly preterm infants, with impaired intestinal defenses and
immature gut barrier function may not be feasible due to the risk
of bacteremia. Therefore, there is a need for compositions that can
provide the benefits of probiotics without introducing viable
bacteria into the intestinal tract of pediatric subjects
[0107] While not wishing to be bound by theory, it is believed that
a culture supernatant from batch cultivation of a probiotic, and in
particular embodiments, LGG, provides beneficial gastrointestinal
benefits. It is further believed that the beneficial effects on gut
barrier function can be attributed to the mixture of components
(including proteinaceous materials, and possibly including
(exo)polysaccharide materials) that are released into the culture
medium at a late stage of the exponential (or "log") phase of batch
cultivation of LGG. The composition will be hereinafter referred to
as "culture supernatant."
[0108] Accordingly, in some embodiments, the nutritional
composition includes a culture supernatant from a late-exponential
growth phase of a probiotic batch-cultivation process. Without
wishing to be bound by theory, it is believed that the activity of
the culture supernatant can be attributed to the mixture of
components (including proteinaceous materials, and possibly
including (exo)polysaccharide materials) as found released into the
culture medium at a late stage of the exponential (or "log") phase
of batch cultivation of the probiotic. The term "culture
supernatant" as used herein, includes the mixture of components
found in the culture medium. The stages recognized in batch
cultivation of bacteria are known to the skilled person. These are
the "lag," the "log" ("logarithmic" or "exponential"), the
"stationary" and the "death" (or "logarithmic decline") phases. In
all phases during which live bacteria are present, the bacteria
metabolize nutrients from the media, and secrete (exert, release)
materials into the culture medium. The composition of the secreted
material at a given point in time of the growth stages is not
generally predictable.
[0109] In an embodiment, a culture supernatant is obtainable by a
process comprising the steps of (a) subjecting a probiotic such as
LGG to cultivation in a suitable culture medium using a batch
process; (b) harvesting the culture supernatant at a late
exponential growth phase of the cultivation step, which phase is
defined with reference to the second half of the time between the
lag phase and the stationary phase of the batch-cultivation
process; (c) optionally removing low molecular weight constituents
from the supernatant so as to retain molecular weight constituents
above 5-6 kiloDaltons (kDa); (d) removing liquid contents from the
culture supernatant so as to obtain the composition.
[0110] The culture supernatant may comprise secreted materials that
are harvested from a late exponential phase. The late exponential
phase occurs in time after the mid exponential phase (which is
halftime of the duration of the exponential phase, hence the
reference to the late exponential phase as being the second half of
the time between the lag phase and the stationary phase). In
particular, the term "late exponential phase" is used herein with
reference to the latter quarter portion of the time between the lag
phase and the stationary phase of the LGG batch-cultivation
process. In some embodiments, the culture supernatant is harvested
at a point in time of 75% to 85% of the duration of the exponential
phase, and may be harvested at about of the time elapsed in the
exponential phase.
[0111] The culture supernatant is believed to contain a mixture of
amino acids, oligo- and polypeptides, and proteins, of various
molecular weights. The composition is further believed to contain
polysaccharide structures and/or nucleotides.
[0112] In some embodiments, the culture supernatant of the present
disclosure excludes low molecular weight components, generally
below 6 kDa, or even below 5 kDa. In these and other embodiments,
the culture supernatant does not include lactic acid and/or lactate
salts. These lower molecular weight components can be removed, for
example, by filtration or column chromatography.
[0113] The culture supernatant of the present disclosure can be
formulated in various ways for administration to pediatric
subjects. For example, the culture supernatant can be used as such,
e.g. incorporated into capsules for oral administration, or in a
liquid nutritional composition such as a drink, or it can be
processed before further use. Such processing generally involves
separating the compounds from the generally liquid continuous phase
of the supernatant. This preferably is done by a drying method,
such as spray-drying or freeze-drying (lyophilization).
Spray-drying is preferred. In a preferred embodiment of the
spray-drying method, a carrier material will be added before
spray-drying, e.g., maltodextrin DE29.
[0114] The LGG culture supernatant of the present disclosure,
whether added in a separate dosage form or via a nutritional
product, will generally be administered in an amount effective in
promoting gut regeneration, promoting gut maturation and/or
protecting gut barrier function. The effective amount is preferably
equivalent to 1.times.10.sup.4 to about 1.times.10.sup.12 cell
equivalents of live probiotic bacteria per kg body weight per day,
and more preferably 10.sup.8-10.sup.9 cell equivalents per kg body
weight per day. In other embodiments, the amount of cell
equivalents may vary from about 1.times.10.sup.4 to about
1.5.times.10.sup.10 cell equivalents of probiotic(s) per 100 Kcal.
In some embodiments the amount of probiotic cell equivalents may be
from about 1.times.10.sup.6 to about 1.times.10.sup.9 cell
equivalents of probiotic(s) per 100 Kcal nutritional composition.
In certain other embodiments the amount of probiotic cell
equivalents may vary from about 1.times.10.sup.7 to about
1.times.10.sup.8 cell equivalents of probiotic(s) per 100 Kcal of
nutritional composition.
[0115] Without being bound by any theory, the unique combination of
nutrients in the disclosed nutritional composition(s) is believed
to be capable of providing novel and unexpected benefits for
infants and children. Moreover, the benefit of this nutritional
composition is believed to be obtained during infancy, and also by
including it as part of a diverse diet as the child continues to
grow and develop.
[0116] In some embodiments, a soluble mediator preparation is
prepared from the culture supernatant as described below.
Furthermore, preparation of an LGG soluble mediator preparation is
described in US 2013/0251829 and US 2011/0217402, each of which is
incorporated by reference in its entirety.
[0117] In certain embodiments, the soluble mediator preparation is
obtainable by a process comprising the steps of (a) subjecting a
probiotic such as LGG to cultivation in a suitable culture medium
using a batch process; (b) harvesting a culture supernatant at a
late exponential growth phase of the cultivation step, which phase
is defined with reference to the second half of the time between
the lag phase and the stationary phase of the batch-cultivation
process; (c) optionally removing low molecular weight constituents
from the supernatant so as to retain molecular weight constituents
above 5-6 kiloDaltons (kDa); (d) removal of any remaining cells
using 0.22 .mu.m sterile filtration to provide the soluble mediator
preparation; (e) removing liquid contents from the soluble mediator
preparation so as to obtain the composition.
[0118] In certain embodiments, secreted materials are harvested
from a late exponential phase. The late exponential phase occurs in
time after the mid exponential phase (which is halftime of the
duration of the exponential phase, hence the reference to the late
exponential phase as being the second half of the time between the
lag phase and the stationary phase). In particular, the term "late
exponential phase" is used herein with reference to the latter
quarter portion of the time between the lag phase and the
stationary phase of the LGG batch-cultivation process. In a
preferred embodiment of the present disclosure and embodiments
thereof, harvesting of the culture supernatant is at a point in
time of 75% to 85% of the duration of the exponential phase, and
most preferably is at about of the time elapsed in the exponential
phase.
[0119] The term "cultivation" or "culturing" refers to the
propagation of microorganisms, in this case LGG, on or in a
suitable medium. Such a culture medium can be of a variety of
kinds, and is particularly a liquid broth, as customary in the art.
A preferred broth, e.g., is MRS broth as generally used for the
cultivation of lactobacilli. MRS broth generally comprises
polysorbate, acetate, magnesium and manganese, which are known to
act as special growth factors for lactobacilli, as well as a rich
nutrient base. A typical composition comprises (amounts in
g/liter): peptone from casein 10.0; yeast extract 4.0; D(+)-glucose
20.0; dipotassium hydrogen phosphate 2.0; Tween.RTM. 80 1.0;
triammonium citrate 2.0; sodium acetate 5.0; magnesium sulphate
0.2; manganese sulphate 0.04.
[0120] In certain embodiments, the soluble mediator preparation is
incorporated into an infant formula or other nutritional
composition. The harvesting of secreted bacterial products brings
about a problem that the culture media cannot easily be deprived of
undesired components. This specifically relates to nutritional
products for relatively vulnerable subjects, such as infant formula
or clinical nutrition. This problem is not incurred if specific
components from a culture supernatant are first isolated, purified,
and then applied in a nutritional product. However, it is desired
to make use of a more complete culture supernatant. This would
serve to provide a soluble mediator composition better reflecting
the natural action of the probiotic (e.g. LGG).
[0121] Accordingly, it is desired to ensure that the composition
harvested from LGG cultivation does not contain components (as may
present in the culture medium) that are not desired, or generally
accepted, in such formula. With reference to polysorbate regularly
present in MRS broth, media for the culturing of bacteria may
include an emulsifying non-ionic surfactant, e.g. on the basis of
polyethoxylated sorbitan and oleic acid (typically available as
Tween.RTM. polysorbates, such as Tween.RTM. 80). Whilst these
surfactants are frequently found in food products, e.g. ice cream,
and are generally recognized as safe, they are not in all
jurisdictions considered desirable, or even acceptable for use in
nutritional products for relatively vulnerable subjects, such as
infant formula or clinical nutrition.
[0122] Therefore, in some embodiments, a preferred culture medium
of the disclosure is devoid of polysorbates such as Tween 80. In a
preferred embodiment of the disclosure and/or embodiments thereof
the culture medium may comprise an oily ingredient selected from
the group consisting of oleic acid, linseed oil, olive oil, rape
seed oil, sunflower oil and mixtures thereof. It will be understood
that the full benefit of the oily ingredient is attained if the
presence of a polysorbate surfactant is essentially or entirely
avoided.
[0123] More particularly, in certain embodiments, an MRS medium is
devoid of polysorbates. Also preferably medium comprises, in
addition to one or more of the foregoing oils, peptone (typically
0-10 g/L, especially 0.1-10 g/L), yeast extract (typically 4-50
g/L), D(+) glucose (typically 20-70 g/L), dipotassium hydrogen
phosphate (typically 2-4 g/L), sodium acetate trihydrate (typically
4-5 g/L), triammonium citrate (typically 2-4 g/L), magnesium
sulfphate heptahydrate (typically 0.2-0.4 g/L) and/or manganous
sulphate tetrahydrate (typically 0.05-0.08 g/L).
[0124] The culturing is generally performed at a temperature of
20.degree. C. to 45.degree. C., more particularly at 35.degree. C.
to 40.degree. C., and more particularly at 37.degree. C. In some
embodiments, the culture has a neutral pH, such as a pH of between
pH 5 and pH 7, preferably pH 6.
[0125] In some embodiments, the time point during cultivation for
harvesting the culture supernatant, i.e., in the aforementioned
late exponential phase, can be determined, e.g. based on the OD600
nm and glucose concentration. OD600 refers to the optical density
at 600 nm, which is a known density measurement that directly
correlates with the bacterial concentration in the culture
medium.
[0126] The culture supernatant can be harvested by any known
technique for the separation of culture supernatant from a
bacterial culture. Such techniques are known in the art and
include, e.g., centrifugation, filtration, sedimentation, and the
like. In some embodiments, LGG cells are removed from the culture
supernatant using 0.22 .mu.m sterile filtration in order to produce
the soluble mediator preparation. The probiotic soluble mediator
preparation thus obtained may be used immediately, or be stored for
future use. In the latter case, the probiotic soluble mediator
preparation will generally be refrigerated, frozen or lyophilized.
The probiotic soluble mediator preparation may be concentrated or
diluted, as desired.
[0127] The soluble mediator preparation is believed to contain a
mixture of amino acids, oligo- and polypeptides, and proteins, of
various molecular weights. The composition is further believed to
contain polysaccharide structures and/or nucleotides.
[0128] In some embodiments, the soluble mediator preparation of the
present disclosure excludes lower molecular weight components,
generally below 6 kDa, or even below 5 kDa. In these and other
embodiments, the soluble mediator preparation does not include
lactic acid and/or lactate salts. These lower molecular weight
components can be removed, for example, by filtration or column
chromatography. In some embodiments, the culture supernatant is
subjected to ultrafiltration with a 5 kDa membrane in order to
retain constituents over 5 kDa. In other embodiments, the culture
supernatant is desalted using column chromatography to retain
constituents over 6 kDa.
[0129] The soluble mediator preparation of the present disclosure
can be formulated in various ways for administration to pediatric
subjects. For example, the soluble mediator preparation can be used
as such, e.g. incorporated into capsules for oral administration,
or in a liquid nutritional composition such as a drink, or it can
be processed before further use. Such processing generally involves
separating the compounds from the generally liquid continuous phase
of the supernatant. This preferably is done by a drying method,
such as spray-drying or freeze-drying (lyophilization). In a
preferred embodiment of the spray-drying method, a carrier material
will be added before spray-drying, e.g., maltodextrin DE29.
[0130] Probiotic bacteria soluble mediator preparations, such as
the LGG soluble mediator preparation disclosed herein,
advantageously possess gut barrier enhancing activity by promoting
gut barrier regeneration, gut barrier maturation and/or adaptation,
gut barrier resistance and/or gut barrier function. The present LGG
soluble mediator preparation may accordingly be particularly useful
in treating subjects, particularly pediatric subjects, with
impaired gut barrier function, such as short bowel syndrome or NEC.
The soluble mediator preparation may be particularly useful for
infants and premature infants having impaired gut barrier function
and/or short bowel syndrome.
[0131] Probiotic bacteria soluble mediator preparation, such as the
LGG soluble mediator preparation of the present disclosure, also
advantageously reduce visceral pain sensitivity in subjects,
particularly pediatric subjects experiencing gastrointestinal pain,
food intolerance, allergic or non-allergic inflammation, colic,
IBS, and infections.
[0132] In an embodiment, the nutritional composition may include
prebiotics. In certain embodiments, the nutritional composition
includes prebiotics that may stimulate endogenous butyrate
production. For example, in some embodiments the component for
stimulating endogenous butyrate production comprises a
microbiota-stimulating component that is a prebiotic including both
polydextrose ("PDX") and galacto-oligosaccharides ("GOS"). A
prebiotic component including PDX and GOS can enhance butyrate
production by microbiota.
[0133] In addition to PDX and GOS, the nutritional composition may
also contain one or more other prebiotics which can exert
additional health benefits, which may include, but are not limited
to, selective stimulation of the growth and/or activity of one or a
limited number of beneficial gut bacteria, stimulation of the
growth and/or activity of ingested probiotic microorganisms,
selective reduction in gut pathogens, and favorable influence on
gut short chain fatty acid profile. Such prebiotics may be
naturally-occurring, synthetic, or developed through the genetic
manipulation of organisms and/or plants, whether such new source is
now known or developed later. Prebiotics useful in the present
disclosure may include oligosaccharides, polysaccharides, and other
prebiotics that contain fructose, xylose, soya, galactose, glucose
and mannose.
[0134] More specifically, prebiotics useful in the present
disclosure include PDX and GOS, and can, in some embodiments, also
include, PDX powder, lactulose, lactosucrose, raffinose,
gluco-oligosaccharide, inulin, fructo-oligosaccharide (FOS),
isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose,
xylo-oligosaccharide (XOS), chito-oligosaccharide,
manno-oligosaccharide, aribino-oligosaccharide,
siallyl-oligosaccharide, fuco-oligosaccharide, and
gentio-oligosaccharides.
[0135] In an embodiment, the total amount of prebiotics present in
the nutritional composition may be from about 1.0 g/L to about 10.0
g/L of the composition. More preferably, the total amount of
prebiotics present in the nutritional composition may be from about
2.0 g/L and about 8.0 g/L of the composition. In some embodiments,
the total amount of prebiotics present in the nutritional
composition may be from about 0.01 g/100 Kcal to about 1.5 g/100
Kcal. In certain embodiments, the total amount of prebiotics
present in the nutritional composition may be from about 0.15 g/100
Kcal to about 1.5 g/100 Kcal. In some embodiments, the prebiotic
component comprises at least 20% w/w PDX and GOS.
[0136] The amount of PDX in the nutritional composition may, in an
embodiment, be within the range of from about 0.015 g/100 Kcal to
about 1.5 g/100 Kcal. In another embodiment, the amount of
polydextrose is within the range of from about 0.2 g/100 Kcal to
about 0.6 g/100 Kcal. In some embodiments, PDX may be included in
the nutritional composition in an amount sufficient to provide
between about 1.0 g/L and 10.0 g/L. In another embodiment, the
nutritional composition contains an amount of PDX that is between
about 2.0 g/L and 8.0 g/L. And in still other embodiments, the
amount of PDX in the nutritional composition may be from about 0.05
g/100 Kcal to about 1.5 g/100 Kcal.
[0137] The prebiotic component also comprises GOS. The amount of
GOS in the nutritional composition may, in an embodiment, be from
about 0.015 g/100 Kcal to about 1.0 g/100 Kcal. In another
embodiment, the amount of GOS in the nutritional composition may be
from about 0.2 g/100 Kcal to about 0.5 g/100 Kcal.
[0138] In a particular embodiment, GOS and PDX are supplemented
into the nutritional composition in a total amount of at least
about 0.015 g/100 Kcal or about 0.015 g/100 Kcal to about 1.5 g/100
Kcal. In some embodiments, the nutritional composition may comprise
GOS and PDX in a total amount of from about 0.1 to about 1.0 g/100
Kcal.
[0139] In some embodiments, the nutritional composition includes a
protein equivalent source, wherein the protein equivalent source
includes a peptide component comprising SEQ ID NO 4, SEQ ID NO 13,
SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 30, SEQ ID NO
31, SEQ ID NO 32, SEQ ID NO 51, SEQ ID NO 57, SEQ ID NO 60, and SEQ
ID NO 63. In some embodiments, the peptide component may comprise
additional peptides disclosed in Table 3. For example, the
composition may include at least 10 additional peptides disclosed
in Table 3. In some embodiments, 20% to 80% of the protein
equivalent source comprises the peptide component, and 20% to 80%
of the protein equivalent source comprises an intact protein, a
partially hydrolyzed protein, and combinations thereof. In some
embodiments, the term additional means selecting different peptides
than those enumerated.
[0140] In another embodiment, 1% to about 99% of the protein
equivalent source includes a peptide component comprising at least
3 peptides selected from the group consisting of SEQ ID NO 4, SEQ
ID NO 13, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 30,
SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 51, SEQ ID NO 57, SEQ ID NO
60, and SEQ ID NO 63, and at least 5 additional peptides selected
from Table 3; and wherein 1% to 99% of the protein equivalent
source comprises an intact protein, a partially hydrolyzed protein,
or combinations thereof. In some embodiments, 20% to 80% of the
protein equivalent source includes a peptide component comprising
at least 3 peptides selected from the group consisting of SEQ ID NO
4, SEQ ID NO 13, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 24, SEQ ID
NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 51, SEQ ID NO 57, SEQ
ID NO 60, and SEQ ID NO 63, and at least 5 additional peptides
selected from Table 3; and wherein 20% to 80% of the protein
equivalent source comprises an intact protein, a partially
hydrolyzed protein, or combinations thereof.
[0141] Table 3 below identifies the amino acid sequences of the
peptides that may be included in the peptide component of the
present nutritional compositions.
TABLE-US-00003 TABLE 3 Seq. ID Amino Acid Sequence (aa) 1 Ala Ile
Asn Pro Ser Lys Glu Asn 8 2 Ala Pro Phe Pro Glu 5 3 Asp Ile Gly Ser
Glu Ser 6 4 Asp Lys Thr Glu Ile Pro Thr 7 5 Asp Met Glu Ser Thr 5 6
Asp Met Pro Ile 4 7 Asp Val Pro Ser 4 n/a Glu Asp Ile 3 n/a Glu Leu
Phe 3 n/a Glu Met Pro 3 8 Glu Thr Ala Pro Val Pro Leu 7 9 Phe Pro
Gly Pro Ile Pro 6 10 Phe Pro Gly Pro Ile Pro Asn 7 11 Gly Pro Phe
Pro 4 12 Gly Pro Ile Val 4 13 Ile Gly Ser Glu Ser Thr Glu Asp Gln 9
14 Ile Gly Ser Ser Ser Glu Glu Ser 8 15 Ile Gly Ser Ser Ser Glu Glu
Ser Ala 9 16 Ile Asn Pro Ser Lys Glu 6 17 Ile Pro Asn Pro Ile 5 18
Ile Pro Asn Pro Ile Gly 6 19 Ile Pro Pro Leu Thr Gln Thr Pro Val 9
20 Ile Thr Ala Pro 4 21 Ile Val Pro Asn 4 22 Lys His Gln Gly Leu
Pro Gln 7 23 Leu Asp Val Thr Pro 5 24 Leu Glu Asp Ser Pro Glu 6 25
Leu Pro Leu Pro Leu 5 26 Met Glu Ser Thr Glu Val 6 27 Met His Gln
Pro His Gln Pro Leu Pro Pro Thr 11 28 Asn Ala Val Pro Ile 5 29 Asn
Glu Val Glu Ala 5 n/a Asn Leu Leu 3 30 Asn Gln Glu Gln Pro Ile 6 31
Asn Val Pro Gly Glu 5 32 Pro Phe Pro Gly Pro Ile 6 33 Pro Gly Pro
Ile Pro Asn 6 34 Pro His Gln Pro Leu Pro Pro Thr 8 35 Pro Ile Thr
Pro Thr 5 36 Pro Asn Pro Ile 4 37 Pro Asn Ser Leu Pro Gln 6 38 Pro
Gln Leu Glu Ile Val Pro Asn 8 39 Pro Gln Asn Ile Pro Pro Leu 7 40
Pro Val Leu Gly Pro Val 6 41 Pro Val Pro Gln 4 42 Pro Val Val Val
Pro 5 43 Pro Val Val Val Pro Pro 6 44 Ser Ile Gly Ser Ser Ser Glu
Glu Ser Ala Glu 11 45 Ser Ile Ser Ser Ser Glu Glu 7 46 Ser Ile Ser
Ser Ser Glu Glu Ile Val Pro Asn 11 47 Ser Lys Asp Ile Gly Ser Glu 7
48 Ser Pro Pro Glu Ile Asn 6 49 Ser Pro Pro Glu Ile Asn Thr 7 50
Thr Asp Ala Pro Ser Phe Ser 7 51 Thr Glu Asp Glu Leu 5 52 Val Ala
Thr Glu Glu Val 6 53 Val Leu Pro Val Pro 5 54 Val Pro Gly Glu 4 55
Val Pro Gly Glu Ile Val 6 56 Val Pro Ile Thr Pro Thr 6 57 Val Pro
Ser Glu 4 58 Val Val Pro Pro Phe Leu Gln Pro Glu 9 59 Val Val Val
Pro Pro 5 60 Tyr Pro Phe Pro Gly Pro 6 61 Tyr Pro Phe Pro Gly Pro
Ile Pro 8 62 Tyr Pro Phe Pro Gly Pro Ile Pro Asn 9 63 Tyr Pro Ser
Gly Ala 5 64 Tyr Pro Val Glu Pro 5
[0142] Table 4 below further identifies a subset of amino acid
sequences from Table 3 that may be included in the peptide
component disclosed herein.
TABLE-US-00004 TABLE 4 Seq ID Number Amino Acid Sequence (aa) 4 Asp
Lys Thr Glu Ile Pro Thr 7 13 Ile Gly Ser Glu Ser Thr Glu Asp Gln 9
17 Ile Pro Asn Pro Ile Gly 6 21 Ile Val Pro Asn 4 24 Leu Glu Asp
Ser Pro Glu 6 30 Asn Gln Glu Gln Pro Ile 6 31 Asn Val Pro Gly Glu 5
32 Pro Phe Pro Gly Pro Ile 6 51 Thr Glu Asp Glu Leu 5 57 Val Pro
Ser Glu 4 60 Tyr Pro Phe Pro Gly Pro 6 63 Tyr Pro Ser Gly Ala 5
[0143] In some embodiments, the peptide component may be present in
the nutritional composition in an amount from about 0.2 g/100 Kcal
to about 5.6 g/100 Kcal. In other embodiments the peptide component
may be present in the nutritional composition in an amount from
about 1 g/100 Kcal to about 4 g/100 Kcal. In still other
embodiments, the peptide component may be present in the
nutritional composition in an amount from about 2 g/100 Kcal to
about 3 g/100 Kcal.
[0144] The peptide component disclosed herein may be formulated
with other ingredients in the nutritional composition to provide
appropriate nutrient levels for the target subject. In some
embodiments, the peptide component is included in a nutritionally
complete formula that is suitable to support normal growth.
[0145] The peptide component may be provided as an element of a
protein equivalent source. In some embodiments, the peptides
identified in Tables 3 and 4, may be provided by a protein
equivalent source obtained from cow's milk proteins, including but
not limited to bovine casein and bovine whey. In some embodiments,
the protein equivalent source comprises hydrolyzed bovine casein or
hydrolyzed bovine whey. Accordingly, in some embodiments, the
peptides identified in Table 3 and Table 4 may be provided by a
casein hydrolysate. Such peptides may be obtained by hydrolysis or
may be synthesized in vitro by methods know to the skilled
person.
[0146] A non-limiting example of a method of hydrolysis is
disclosed herein. In some embodiments, this method may be used to
obtain the protein hydrolysate and peptides of the present
disclosure. The proteins are hydrolyzed using a proteolytic enzyme,
Protease N. Protease N "Amano" is commercially available from Amano
Enzyme U.S.A. Co., Ltd., Elgin, Ill. Protease N is a proteolytic
enzyme preparation that is derived from the bacterial species
Bacillus subtilis. The protease powder is specified as "not less
than 150,000 units/g", meaning that one unit of Protease N is the
amount of enzyme which produces an amino acid equivalent to 100
micrograms of tyrosine for 60 minutes at a pH of 7.0. To produce
the infant formula of the present disclosure, Protease N can be
used at levels of about 0.5% to about 1.0% by weight of the total
protein being hydrolyzed.
[0147] The protein hydrolysis by Protease N is typically conducted
at a temperature of about 50.degree. C. to about 60.degree. C. The
hydrolysis occurs for a period of time so as to obtain a degree of
hydrolysis between about 4% and 10%. In a particular embodiment,
hydrolysis occurs for a period of time so as to obtain a degree of
hydrolysis between about 6% and 9%. In another embodiment,
hydrolysis occurs for a period of time so as to obtain a degree of
hydrolysis of about 7.5%. This level of hydrolysis may take between
about one half hour to about 3 hours.
[0148] A constant pH should be maintained during hydrolysis. In the
method of the present disclosure, the pH is adjusted to and
maintained between about 6.5 and 8. In a particular embodiment, the
pH is maintained at about 7.0.
[0149] In order to maintain the optimal pH of the solution of whey
protein, casein, water and Protease N, a caustic solution of sodium
hydroxide and/or potassium hydroxide can be used to adjust the pH
during hydrolysis. If sodium hydroxide is used to adjust the pH,
the amount of sodium hydroxide added to the solution should be
controlled to the level that it comprises less than about 0.3% of
the total solid in the finished protein hydrolysate. A 10%
potassium hydroxide solution can also be used to adjust the pH of
the solution to the desired value, either before the enzyme is
added or during the hydrolysis process in order to maintain the
optimal pH.
[0150] The amount of caustic solution added to the solution during
the protein hydrolysis can be controlled by a pH-stat or by adding
the caustic solution continuously and proportionally. The
hydrolysate can be manufactured by standard batch processes or by
continuous processes.
[0151] To better ensure the consistent quality of the protein
partial hydrolysate, the hydrolysate is subjected to enzyme
deactivation to end the hydrolysis process. The enzyme deactivation
step may consist include at heat treatment at a temperature of
about 82.degree. C. for about 10 minutes. Alternatively, the enzyme
can be deactivated by heating the solution to a temperature of
about 92.degree. C. for about 5 seconds. After enzyme deactivation
is complete, the hydrolysate can be stored in a liquid state at a
temperature lower than 10.degree. C.
[0152] In some embodiments, the protein equivalent source comprises
a hydrolyzed protein, which includes partially hydrolyzed protein
and extensively hydrolyzed protein, such as casein. In some
embodiments, the protein equivalent source comprises a hydrolyzed
protein including peptides having a molar mass distribution of
greater than 500 Daltons. In some embodiments, the hydrolyzed
protein comprises peptides having a molar mass distribution in the
range of from about 500 Daltons to about 1,500 Daltons. Still, in
some embodiments the hydrolyzed protein may comprise peptides
having a molar mass distribution range of from about 500 Daltons to
about 2,000 Daltons.
[0153] In some embodiments, the protein equivalent source may
comprise the peptide component, intact protein, hydrolyzed protein,
including partially hydrolyzed protein and/or extensively
hydrolyzed protein, and combinations thereof. In some embodiments,
1% to 99% of the protein equivalent source comprises the peptide
component disclosed herein. In some embodiments, 10% to 90% of the
protein equivalent source comprises the peptide component disclosed
herein. In some embodiments, 20% to 80% of the protein equivalent
source comprises the peptide component disclosed herein. In some
embodiments, 30% to 60% of the protein equivalent source comprises
the peptide component disclosed herein. In still other embodiments,
40% to 50% of the protein equivalent source comprises the peptide
component.
[0154] In some embodiments, 1% to 99% of the protein equivalent
source comprises intact protein, partially hydrolyzed protein,
extensively hydrolyzed protein, or combinations thereof. In some
embodiments, 10% to 90% of the protein equivalent source comprises
intact protein, partially hydrolyzed protein, extensively
hydrolyzed protein, or combinations thereof. In some embodiments,
20% to 80% of the protein equivalent source comprises intact
protein, partially hydrolyzed protein, extensively hydrolyzed
protein, or combinations thereof. In some embodiments, 40% to 70%
of the protein equivalent source comprises intact proteins,
partially hydrolyzed proteins, extensively hydrolyzed protein, or a
combination thereof. In still further embodiments, 50% to 60% of
the protein equivalent source may comprise intact proteins,
partially hydrolyzed protein, extensively hydrolyzed protein, or a
combination thereof.
[0155] In some embodiments the protein equivalent source comprises
partially hydrolyzed protein having a degree of hydrolysis of less
than 40%. In still other embodiments, the protein equivalent source
may comprise partially hydrolyzed protein having a degree of
hydrolysis of less than 25%, or less than 15%.
[0156] In some embodiments, the nutritional composition comprises
between about 1 g and about 7 g of a protein equivalent source per
100 Kcal. In other embodiments, the nutritional composition
comprises between about 3.5 g and about 4.5 g of protein equivalent
source per 100 Kcal.
[0157] The nutritional composition(s) of the disclosure may also
comprise a protein source. The protein source can be any used in
the art, e.g., nonfat milk, whey protein, casein, soy protein,
hydrolyzed protein, amino acids, and the like. Bovine milk protein
sources useful in practicing the present disclosure include, but
are not limited to, milk protein powders, milk protein
concentrates, milk protein isolates, nonfat milk solids, nonfat
milk, nonfat dry milk, whey protein, whey protein isolates, whey
protein concentrates, sweet whey, acid whey, casein, acid casein,
caseinate (e.g. sodium caseinate, sodium calcium caseinate, calcium
caseinate) and any combinations thereof.
[0158] In one embodiment, the proteins of the nutritional
composition are provided as intact proteins. In other embodiments,
the proteins are provided as a combination of both intact proteins
and partially hydrolyzed proteins, with a degree of hydrolysis of
between about 4% and 10%. In certain other embodiments, the
proteins are more completely hydrolyzed. In still other
embodiments, the protein source comprises amino acids. In yet
another embodiment, the protein source may be supplemented with
glutamine-containing peptides.
[0159] In a particular embodiment of the nutritional composition,
the whey:casein ratio of the protein source is similar to that
found in human breast milk. In an embodiment, the protein source
comprises from about 40% to about 80% whey protein and from about
20% to about 60% casein.
[0160] In some embodiments the protein source may include a
combination of milk powders and whey protein powders. In some
embodiments, the protein source comprise from about 5 wt % to about
30% of nonfat milk powder based on the total weight of the
nutritional composition and about 2 wt % to about 20 wt % of whey
protein concentrate based on the total weight of the nutritional
composition. Still in certain embodiments, the protein source
comprise from about 10 wt % to about 20% of nonfat milk powder
based on the total weight of the nutritional composition and about
5 wt % to about 15 wt % of whey protein concentrate based on the
total weight of the nutritional composition.
[0161] In some embodiments, the nutritional composition comprises
between about 1 g and about 7 g of a protein source per 100 Kcal.
In other embodiments, the nutritional composition comprises between
about 3.5 g and about 4.5 g of protein per 100 Kcal.
[0162] The nutritional composition(s) of the present disclosure may
also comprise a carbohydrate source. Carbohydrate sources can be
any used in the art, e.g., lactose, glucose, fructose, corn syrup
solids, maltodextrins, sucrose, starch, rice syrup solids, and the
like. The amount of carbohydrate in the nutritional composition
typically can vary from between about 5 g and about 25 g/100 Kcal.
In some embodiments, the amount of carbohydrate is between about 6
g and about 22 g/100 Kcal. In other embodiments, the amount of
carbohydrate is between about 12 g and about 14 g/100 Kcal. In some
embodiments, corn syrup solids are preferred. Moreover, hydrolyzed,
partially hydrolyzed, and/or extensively hydrolyzed carbohydrates
may be desirable for inclusion in the nutritional composition due
to their easy digestibility. Specifically, hydrolyzed carbohydrates
are less likely to contain allergenic epitopes.
[0163] Non-limiting examples of carbohydrate materials suitable for
use herein include hydrolyzed or intact, naturally or chemically
modified, starches sourced from corn, tapioca, rice or potato, in
waxy or non-waxy forms. Non-limiting examples of suitable
carbohydrates include various hydrolyzed starches characterized as
hydrolyzed cornstarch, maltodextrin, maltose, corn syrup, dextrose,
corn syrup solids, glucose, and various other glucose polymers and
combinations thereof. Non-limiting examples of other suitable
carbohydrates include those often referred to as sucrose, lactose,
fructose, high fructose corn syrup, indigestible oligosaccharides
such as fructooligosaccharides and combinations thereof.
[0164] In some embodiments, the nutritional composition described
herein comprises a fat source. The enriched lipid fraction
described herein may be the sole fat source or may be used in
combination with any other suitable fat or lipid source for the
nutritional composition as known in the art. In certain
embodiments, appropriate fat sources include, but are not limited
to, animal sources, e.g., milk fat, butter, butter fat, egg yolk
lipid; marine sources, such as fish oils, marine oils, single cell
oils; vegetable and plant oils, such as corn oil, canola oil,
sunflower oil, soybean oil, palm olein oil, coconut oil, high oleic
sunflower oil, evening primrose oil, rapeseed oil, olive oil,
flaxseed (linseed) oil, cottonseed oil, high oleic safflower oil,
palm stearin, palm kernel oil, wheat germ oil; medium chain
triglyceride oils and emulsions and esters of fatty acids; and any
combinations thereof.
[0165] In some embodiment the nutritional composition comprises
between about 1 g/100 Kcal to about 10 g/100 Kcal of a fat or lipid
source. In some embodiments, the nutritional composition comprises
between about 2 g/100 Kcal to about 7 g/100 Kcal of a fat source.
In other embodiments the fat source may be present in an amount
from about 2.5 g/100 Kcal to about 6 g/100 Kcal. In still other
embodiments, the fat source may be present in the nutritional
composition in an amount from about 3 g/100 Kcal to about 4 g/100
Kcal.
[0166] In some embodiments, the fat or lipid source comprises from
about 10% to about 35% palm oil per the total amount of fat or
lipid. In some embodiments, the fat or lipid source comprises from
about 15% to about 30% palm oil per the total amount of fat or
lipid. Yet in other embodiments, the fat or lipid source may
comprise from about 18% to about 25% palm oil per the total amount
of fat or lipid.
[0167] In certain embodiments, the fat or lipid source may be
formulated to include from about 2% to about 16% soybean oil based
on the total amount of fat or lipid. In some embodiments, the fat
or lipid source may be formulated to include from about 4% to about
12% soybean oil based on the total amount of fat or lipid. In some
embodiments, the fat or lipid source may be formulated to include
from about 6% to about 10% soybean oil based on the total amount of
fat or lipid.
[0168] In certain embodiments, the fat or lipid source may be
formulated to include from about 2% to about 16% coconut oil based
on the total amount of fat or lipid. In some embodiments, the fat
or lipid source may be formulated to include from about 4% to about
12% coconut oil based on the total amount of fat or lipid. In some
embodiments, the fat or lipid source may be formulated to include
from about 6% to about 10% coconut oil based on the total amount of
fat or lipid.
[0169] In certain embodiments, the fat or lipid source may be
formulated to include from about 2% to about 16% sunflower oil
based on the total amount of fat or lipid. In some embodiments, the
fat or lipid source may be formulated to include from about 4% to
about 12% sunflower oil based on the total amount of fat or lipid.
In some embodiments, the fat or lipid source may be formulated to
include from about 6% to about 10% sunflower oil based on the total
amount of fat or lipid.
[0170] In some embodiments, the oils, i.e. sunflower oil, soybean
oil, sunflower oil, palm oil, etc. are meant to cover fortified
versions of such oils known in the art. For example, in certain
embodiments, the use of sunflower oil may include high oleic
sunflower oil. In other examples, the use of such oils may be
fortified with certain fatty acids, as known in the art, and may be
used in the fat or lipid source disclosed herein.
[0171] In some embodiments the nutritional composition may also
include a source of LCPUFAs. In one embodiment the amount of LCPUFA
in the nutritional composition is advantageously at least about 5
mg/100 Kcal, and may vary from about 5 mg/100 Kcal to about 100
mg/100 Kcal, more preferably from about 10 mg/100 Kcal to about 50
mg/100 Kcal. Non-limiting examples of LCPUFAs include, but are not
limited to, DHA, ARA, linoleic (18:2 n-6), .gamma.-linolenic (18:3
n-6), dihomo-.gamma.-linolenic (20:3 n-6) acids in the n-6 pathway,
.alpha.-linolenic (18:3 n-3), stearidonic (18:4 n-3),
eicosatetraenoic (20:4 n-3), eicosapentaenoic (20:5 n-3), and
docosapentaenoic (22:6 n-3).
[0172] In some embodiments, the LCPUFA included in the nutritional
composition may comprise DHA. In one embodiment the amount of DHA
in the nutritional composition is advantageously at least about 17
mg/100 Kcal, and may vary from about 5 mg/100 Kcal to about 75
mg/100 Kcal, more preferably from about 10 mg/100 Kcal to about 50
mg/100 Kcal.
[0173] In another embodiment, especially if the nutritional
composition is an infant formula, the nutritional composition is
supplemented with both DHA and ARA. In this embodiment, the weight
ratio of ARA:DHA may be between about 1:3 and about 9:1. In a
particular embodiment, the ratio of ARA:DHA is from about 1:2 to
about 4:1.
[0174] The DHA and ARA can be in natural form, provided that the
remainder of the LCPUFA source does not result in any substantial
deleterious effect on the infant. Alternatively, the DHA and ARA
can be used in refined form.
[0175] The disclosed nutritional composition described herein can,
in some embodiments, also comprise a source of .beta.-glucan.
Glucans are polysaccharides, specifically polymers of glucose,
which are naturally occurring and may be found in cell walls of
bacteria, yeast, fungi, and plants. Beta glucans (.beta.-glucans)
are themselves a diverse subset of glucose polymers, which are made
up of chains of glucose monomers linked together via beta-type
glycosidic bonds to form complex carbohydrates.
[0176] .beta.-1,3-glucans are carbohydrate polymers purified from,
for example, yeast, mushroom, bacteria, algae, or cereals. The
chemical structure of .beta.-1,3-glucan depends on the source of
the .beta.-1,3-glucan. Moreover, various physiochemical parameters,
such as solubility, primary structure, molecular weight, and
branching, play a role in biological activities of
.beta.-1,3-glucans.
[0177] .beta.-1,3-glucans are naturally occurring polysaccharides,
with or without .beta.-1,6-glucose side chains that are found in
the cell walls of a variety of plants, yeasts, fungi and bacteria.
.beta.-1,3;1,6-glucans are those containing glucose units with
(1,3) links having side chains attached at the (1,6) position(s).
.beta.-1,3;1,6 glucans are a heterogeneous group of glucose
polymers that share structural commonalities, including a backbone
of straight chain glucose units linked by a .beta.-1,3 bond with
.beta.-1,6-linked glucose branches extending from this backbone.
While this is the basic structure for the presently described class
of .beta.-glucans, some variations may exist. For example, certain
yeast .beta.-glucans have additional regions of .beta.(1,3)
branching extending from the .beta.(1,6) branches, which add
further complexity to their respective structures.
[0178] .beta.-glucans derived from baker's yeast, Saccharomyces
cerevisiae, are made up of chains of D-glucose molecules connected
at the 1 and 3 positions, having side chains of glucose attached at
the 1 and 6 positions. Yeast-derived .beta.-glucan is an insoluble,
fiber-like, complex sugar having the general structure of a linear
chain of glucose units with a .beta.-1,3 backbone interspersed with
.beta.-1,6 side chains that are generally 6-8 glucose units in
length. More specifically, .beta.-glucan derived from baker's yeast
is poly-(1,6)-8-D-glucopyranosyl-(1,3)-13-D-glucopyranose.
[0179] Furthermore, .beta.-glucans are well tolerated and do not
produce or cause excess gas, abdominal distension, bloating or
diarrhea in pediatric subjects. Addition of .beta.-glucan to a
nutritional composition for a pediatric subject, such as an infant
formula, a growing-up milk or another children's nutritional
product, will improve the subject's immune response by increasing
resistance against invading pathogens and therefore maintaining or
improving overall health.
[0180] In some embodiments, the .beta.-glucan is
.beta.-1,3;1,6-glucan. In some embodiments, the
.beta.-1,3;1,6-glucan is derived from baker's yeast. The
nutritional composition may comprise whole glucan particle
.beta.-glucan, particulate .delta.-glucan, PGG-glucan
(poly-1,6-.beta.-D-glucopyranosyl-1,3-.beta.-D-glucopyranose) or
any mixture thereof.
[0181] In some embodiments, the amount of .beta.-glucan in the
nutritional composition is between about 3 mg and about 17 mg per
100 Kcal. In another embodiment the amount of .beta.-glucan is
between about 6 mg and about 17 mg per 100 Kcal.
[0182] The nutritional composition of the present disclosure may
comprise lactoferrin in some embodiments. Lactoferrins are single
chain polypeptides of about 80 kD containing 1-4 glycans, depending
on the species. The 3-D structures of lactoferrin of different
species are very similar, but not identical. Each lactoferrin
comprises two homologous lobes, called the N- and C-lobes,
referring to the N-terminal and C-terminal part of the molecule,
respectively. Each lobe further consists of two sub-lobes or
domains, which form a cleft where the ferric ion (Fe3+) is tightly
bound in synergistic cooperation with a (bi)carbonate anion. These
domains are called N1, N2, C1 and C2, respectively. The N-terminus
of lactoferrin has strong cationic peptide regions that are
responsible for a number of important binding characteristics.
Lactoferrin has a very high isoelectric point (.about.pl 9) and its
cationic nature plays a major role in its ability to defend against
bacterial, viral, and fungal pathogens. There are several clusters
of cationic amino acids residues within the N-terminal region of
lactoferrin mediating the biological activities of lactoferrin
against a wide range of microorganisms.
[0183] Lactoferrin for use in the present disclosure may be, for
example, isolated from the milk of a non-human animal or produced
by a genetically modified organism. The oral electrolyte solutions
described herein can, in some embodiments comprise non-human
lactoferrin, non-human lactoferrin produced by a genetically
modified organism and/or human lactoferrin produced by a
genetically modified organism.
[0184] Suitable non-human lactoferrins for use in the present
disclosure include, but are not limited to, those having at least
48% homology with the amino acid sequence of human lactoferrin. For
instance, bovine lactoferrin (bLF) has an amino acid composition
which has about 70% sequence homology to that of human lactoferrin.
In some embodiments, the non-human lactoferrin has at least 65%
homology with human lactoferrin and in some embodiments, at least
75% homology. Non-human lactoferrins acceptable for use in the
present disclosure include, without limitation, bLF, porcine
lactoferrin, equine lactoferrin, buffalo lactoferrin, goat
lactoferrin, murine lactoferrin and camel lactoferrin.
[0185] In some embodiments, the nutritional composition of the
present disclosure comprises non-human lactoferrin, for example
bLF. bLF is a glycoprotein that belongs to the iron transporter or
transferring family. It is isolated from bovine milk, wherein it is
found as a component of whey. There are known differences between
the amino acid sequence, glycosylation patters and iron-binding
capacity in human lactoferrin and bLF. Additionally, there are
multiple and sequential processing steps involved in the isolation
of bLF from cow's milk that affect the physiochemical properties of
the resulting bLF preparation. Human lactoferrin and bLF are also
reported to have differences in their abilities to bind the
lactoferrin receptor found in the human intestine.
[0186] Though not wishing to be bound by this or any other theory,
it is believe that bLF that has been isolated from whole milk has
less lipopolysaccharide (LPS) initially bound than does bLF that
has been isolated from milk powder. Additionally, it is believed
that bLF with a low somatic cell count has less initially-bound
LPS. A bLF with less initially-bound LPS has more binding sites
available on its surface. This is thought to aid bLF in binding to
the appropriate location and disrupting the infection process.
[0187] bLF suitable for the present disclosure may be produced by
any method known in the art. For example, in U.S. Pat. No.
4,791,193, incorporated by reference herein in its entirety,
Okonogi et al. discloses a process for producing bovine lactoferrin
in high purity. Generally, the process as disclosed includes three
steps. Raw milk material is first contacted with a weakly acidic
cationic exchanger to absorb lactoferrin followed by the second
step where washing takes place to remove nonabsorbed substances. A
desorbing step follows where lactoferrin is removed to produce
purified bovine lactoferrin. Other methods may include steps as
described in U.S. Pat. Nos. 7,368,141, 5,849,885, 5,919,913 and
5,861,491, the disclosures of which are all incorporated by
reference in their entirety.
[0188] In certain embodiments, lactoferrin utilized in the present
disclosure may be provided by an expanded bed absorption (EBA)
process for isolating proteins from milk sources. EBA, also
sometimes called stabilized fluid bed adsorption, is a process for
isolating a milk protein, such as lactoferrin, from a milk source
comprises establishing an expanded bed adsorption column comprising
a particulate matrix, applying a milk source to the matrix, and
eluting the lactoferrin from the matrix with an elution buffer
comprising about 0.3 to about 2.0 M sodium chloride. Any mammalian
milk source may be used in the present processes, although in
particular embodiments, the milk source is a bovine milk source.
The milk source comprises, in some embodiments, whole milk, reduced
fat milk, skim milk, whey, casein, or mixtures thereof.
[0189] In particular embodiments, the target protein is
lactoferrin, though other milk proteins, such as lactoperoxidases
or lactalbumins, also may be isolated. In some embodiments, the
process comprises the steps of establishing an expanded bed
adsorption column comprising a particulate matrix, applying a milk
source to the matrix, and eluting the lactoferrin from the matrix
with about 0.3 to about 2.0M sodium chloride. In other embodiments,
the lactoferrin is eluted with about 0.5 to about 1.0 M sodium
chloride, while in further embodiments, the lactoferrin is eluted
with about 0.7 to about 0.9 M sodium chloride.
[0190] The expanded bed adsorption column can be any known in the
art, such as those described in U.S. Pat. Nos. 7,812,138,
6,620,326, and 6,977,046, the disclosures of which are hereby
incorporated by reference herein. In some embodiments, a milk
source is applied to the column in an expanded mode, and the
elution is performed in either expanded or packed mode. In
particular embodiments, the elution is performed in an expanded
mode. For example, the expansion ratio in the expanded mode may be
about 1 to about 3, or about 1.3 to about 1.7. EBA technology is
further described in international published application nos. WO
92/00799, WO 02/18237, WO 97/17132, which are hereby incorporated
by reference in their entireties.
[0191] The isoelectric point of lactoferrin is approximately 8.9.
Prior EBA methods of isolating lactoferrin use 200 mM sodium
hydroxide as an elution buffer. Thus, the pH of the system rises to
over 12, and the structure and bioactivity of lactoferrin may be
comprised, by irreversible structural changes. It has now been
discovered that a sodium chloride solution can be used as an
elution buffer in the isolation of lactoferrin from the EBA matrix.
In certain embodiments, the sodium chloride has a concentration of
about 0.3 M to about 2.0 M. In other embodiments, the lactoferrin
elution buffer has a sodium chloride concentration of about 0.3 M
to about 1.5 M, or about 0.5 m to about 1.0 M.
[0192] In other embodiments, lactoferrin for use in the composition
of the present disclosure can be isolated through the use of radial
chromatography or charged membranes, as would be familiar to the
skilled artisan.
[0193] The lactoferrin that is used in certain embodiments may be
any lactoferrin isolated from whole milk and/or having a low
somatic cell count, wherein "low somatic cell count" refers to a
somatic cell count less than 200,000 cells/mL. By way of example,
suitable lactoferrin is available from Tatua Co-operative Dairy Co.
Ltd., in Morrinsville, New Zealand, from FrieslandCampina Domo in
Amersfoort, Netherlands or from Fonterra Co-Operative Group Limited
in Auckland, New Zealand.
[0194] Surprisingly, lactoferrin included herein maintains certain
bactericidal activity even if exposed to a low pH (i.e., below
about 7, and even as low as about 4.6 or lower) and/or high
temperatures (i.e., above about 65.degree. C., and as high as about
120.degree. C.), conditions which would be expected to destroy or
severely limit the stability or activity of human lactoferrin.
These low pH and/or high temperature conditions can be expected
during certain processing regimen for nutritional compositions of
the types described herein, such as pasteurization. Therefore, even
after processing regimens, lactoferrin has bactericidal activity
against undesirable bacterial pathogens found in the human gut. The
nutritional composition may, in some embodiments, comprise
lactoferrin in an amount from about 25 mg/100 mL to about 150
mg/100 mL. In other embodiments lactoferrin is present in an amount
from about 60 mg/100 mL to about 120 mg/100 mL. In still other
embodiments lactoferrin is present in an amount from about 85
mg/100 mL to about 110 mg/100 mL.
[0195] The disclosed nutritional composition described herein, can,
in some embodiments also comprise an effective amount of iron. The
iron may comprise encapsulated iron forms, such as encapsulated
ferrous fumarate or encapsulated ferrous sulfate or less reactive
iron forms, such as ferric pyrophosphate or ferric
orthophosphate.
[0196] One or more vitamins and/or minerals may also be added in to
the nutritional composition in amounts sufficient to supply the
daily nutritional requirements of a subject. It is to be understood
by one of ordinary skill in the art that vitamin and mineral
requirements will vary, for example, based on the age of the child.
For instance, an infant may have different vitamin and mineral
requirements than a child between the ages of one and thirteen
years. Thus, the embodiments are not intended to limit the
nutritional composition to a particular age group but, rather, to
provide a range of acceptable vitamin and mineral components.
[0197] In embodiments providing a nutritional composition for a
child, the composition may optionally include, but is not limited
to, one or more of the following vitamins or derivations thereof:
vitamin B.sub.1 (thiamin, thiamin pyrophosphate, TPP, thiamin
triphosphate, TTP, thiamin hydrochloride, thiamin mononitrate),
vitamin B.sub.2 (riboflavin, flavin mononucleotide, FMN, flavin
adenine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin B.sub.3
(niacin, nicotinic acid, nicotinamide, niacinamide, nicotinamide
adenine dinucleotide, NAD, nicotinic acid mononucleotide, NicMN,
pyridine-3-carboxylic acid), vitamin B.sub.3-precursor tryptophan,
vitamin B.sub.6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxine
hydrochloride), pantothenic acid (pantothenate, panthenol), folate
(folic acid, folacin, pteroylglutamic acid), vitamin B.sub.12
(cobalamin, methylcobalamin, deoxyadenosylcobalamin,
cyanocobalamin, hydroxycobalamin, adenosylcobalamin), biotin,
vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate,
retinyl palmitate, retinyl esters with other long-chain fatty
acids, retinal, retinoic acid, retinol esters), vitamin D
(calciferol, cholecalciferol, vitamin D.sub.3,
1,25,-dihydroxyvitamin D), vitamin E (.alpha.-tocopherol,
.alpha.-tocopherol acetate, .alpha.-tocopherol succinate,
.alpha.-tocopherol nicotinate, .alpha.-tocopherol), vitamin K
(vitamin K.sub.1, phylloquinone, naphthoquinone, vitamin K.sub.2,
menaquinone-7, vitamin K.sub.3, menaquinone-4, menadione,
menaquinone-8, menaquinone-8H, menaquinone-9, menaquinone-9H,
menaquinone-10, menaquinone-11, menaquinone-12, menaquinone-13),
choline, inositol, .beta.-carotene and any combinations
thereof.
[0198] In embodiments providing a children's nutritional product,
such as a growing-up milk, the composition may optionally include,
but is not limited to, one or more of the following minerals or
derivations thereof: boron, calcium, calcium acetate, calcium
gluconate, calcium chloride, calcium lactate, calcium phosphate,
calcium sulfate, chloride, chromium, chromium chloride, chromium
picolonate, copper, copper sulfate, copper gluconate, cupric
sulfate, fluoride, iron, carbonyl iron, ferric iron, ferrous
fumarate, ferric orthophosphate, iron trituration, polysaccharide
iron, iodide, iodine, magnesium, magnesium carbonate, magnesium
hydroxide, magnesium oxide, magnesium stearate, magnesium sulfate,
manganese, molybdenum, phosphorus, potassium, potassium phosphate,
potassium iodide, potassium chloride, potassium acetate, selenium,
sulfur, sodium, docusate sodium, sodium chloride, sodium selenate,
sodium molybdate, zinc, zinc oxide, zinc sulfate and mixtures
thereof. Non-limiting exemplary derivatives of mineral compounds
include salts, alkaline salts, esters and chelates of any mineral
compound.
[0199] The minerals can be added to growing-up milks or to other
children's nutritional compositions in the form of salts such as
calcium phosphate, calcium glycerol phosphate, sodium citrate,
potassium chloride, potassium phosphate, magnesium phosphate,
ferrous sulfate, zinc sulfate, cupric sulfate, manganese sulfate,
and sodium selenite. Additional vitamins and minerals can be added
as known within the art.
[0200] The nutritional compositions of the present disclosure may
optionally include one or more of the following flavoring agents,
including, but not limited to, flavored extracts, volatile oils,
cocoa or chocolate flavorings, peanut butter flavoring, cookie
crumbs, vanilla or any commercially available flavoring. Examples
of useful flavorings include, but are not limited to, pure anise
extract, imitation banana extract, imitation cherry extract,
chocolate extract, pure lemon extract, pure orange extract, pure
peppermint extract, honey, imitation pineapple extract, imitation
rum extract, imitation strawberry extract, or vanilla extract; or
volatile oils, such as balm oil, bay oil, bergamot oil, cedarwood
oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut
butter, chocolate flavoring, vanilla cookie crumb, butterscotch,
toffee, and mixtures thereof. The amounts of flavoring agent can
vary greatly depending upon the flavoring agent used. The type and
amount of flavoring agent can be selected as is known in the
art.
[0201] The nutritional compositions of the present disclosure may
optionally include one or more emulsifiers that may be added for
stability of the final product. Examples of suitable emulsifiers
include, but are not limited to, lecithin (e.g., from egg or soy),
alpha lactalbumin and/or mono- and di-glycerides, and mixtures
thereof. Other emulsifiers are readily apparent to the skilled
artisan and selection of suitable emulsifier(s) will depend, in
part, upon the formulation and final product. Indeed, the
incorporation of dietary butyrate into a nutritional composition,
such as an infant formula, may require the presence of at least on
emulsifier to ensure that the dietary butyrate does not separate
from the fat or proteins contained within the infant formula during
shelf-storage or preparation.
[0202] In some embodiments, the nutritional composition may be
formulated to include from about 0.5 wt % to about 1 wt % of
emulsifier based on the total dry weight of the nutritional
composition. In other embodiments, the nutritional composition may
be formulated to include from about 0.7 wt % to about 1 wt % of
emulsifier based on the total dry weight of the nutritional
composition.
[0203] In some embodiments where the nutritional composition is a
ready-to-use liquid composition, the nutritional composition may be
formulated to include from about 200 mg/L to about 600 mg/L of
emulsifier. Still, in certain embodiments, the nutritional
composition may include from about 300 mg/L to about 500 mg/L of
emulsifier. In other embodiments, the nutritional composition may
include from about 400 mg/L to about 500 mg/L of emulsifier.
[0204] The nutritional compositions of the present disclosure may
optionally include one or more preservatives that may also be added
to extend product shelf life. Suitable preservatives include, but
are not limited to, potassium sorbate, sodium sorbate, potassium
benzoate, sodium benzoate, potassium citrate, calcium disodium
EDTA, and mixtures thereof. The incorporation of a preservative in
the nutritional composition including dietary butyrate ensures that
the nutritional composition has a suitable shelf-life such that,
once reconstituted for administration, the nutritional composition
delivers nutrients that are bioavailable and/or provide health and
nutrition benefits for the target subject.
[0205] In some embodiments the nutritional composition may be
formulated to include from about 0.1 wt % to about 1.0 wt % of a
preservative based on the total dry weight of the composition. In
other embodiments, the nutritional composition may be formulated to
include from about 0.4 wt % to about 0.7 wt % of a preservative
based on the total dry weight of the composition.
[0206] In some embodiments where the nutritional composition is a
ready-to-use liquid composition, the nutritional composition may be
formulated to include from about 0.5 g/L to about 5 g/L of
preservative. Still, in certain embodiments, the nutritional
composition may include from about 1 g/L to about 3 g/L of
preservative.
[0207] The nutritional compositions of the present disclosure may
optionally include one or more stabilizers. Suitable stabilizers
for use in practicing the nutritional composition of the present
disclosure include, but are not limited to, gum arabic, gum ghatti,
gum karaya, gum tragacanth, agar, furcellaran, guar gum, gellan
gum, locust bean gum, pectin, low methoxyl pectin, gelatin,
microcrystalline cellulose, CMC (sodium carboxymethylcellulose),
methylcellulose hydroxypropyl methyl cellulose, hydroxypropyl
cellulose, DATEM (diacetyl tartaric acid esters of mono- and
diglycerides), dextran, carrageenans, and mixtures thereof. Indeed,
incorporating a suitable stabilizer in the nutritional composition
including dietary butyrate ensures that the nutritional composition
has a suitable shelf-life such that, once reconstituted for
administration, the nutritional composition delivers nutrients that
are bioavailable and/or provide health and nutrition benefits for
the target subject.
[0208] In some embodiments where the nutritional composition is a
ready-to-use liquid composition, the nutritional composition may be
formulated to include from about 50 mg/L to about 150 mg/L of
stabilizer. Still, in certain embodiments, the nutritional
composition may include from about 80 mg/L to about 120 mg/L of
stabilizer.
[0209] The nutritional compositions of the disclosure may provide
minimal, partial or total nutritional support. The compositions may
be nutritional supplements or meal replacements. The compositions
may, but need not, be nutritionally complete. In an embodiment, the
nutritional composition of the disclosure is nutritionally complete
and contains suitable types and amounts of lipid, carbohydrate,
protein, vitamins and minerals. The amount of lipid or fat
typically can vary from about 1 to about 25 g/100 Kcal. The amount
of protein typically can vary from about 1 to about 7 g/100 Kcal.
The amount of carbohydrate typically can vary from about 6 to about
22 g/100 Kcal.
[0210] In an embodiment, the children's nutritional composition may
contain between about 10 and about 50% of the maximum dietary
recommendation for any given country, or between about 10 and about
50% of the average dietary recommendation for a group of countries,
per serving of vitamins A, C, and E, zinc, iron, iodine, selenium,
and choline. In another embodiment, the children's nutritional
composition may supply about 10-30% of the maximum dietary
recommendation for any given country, or about 10-30% of the
average dietary recommendation for a group of countries, per
serving of B-vitamins. In yet another embodiment, the levels of
vitamin D, calcium, magnesium, phosphorus, and potassium in the
children's nutritional product may correspond with the average
levels found in milk. In other embodiments, other nutrients in the
children's nutritional composition may be present at about 20% of
the maximum dietary recommendation for any given country, or about
20% of the average dietary recommendation for a group of countries,
per serving.
[0211] In some embodiments the nutritional composition is an infant
formula. Infant formulas are fortified nutritional compositions for
an infant. The content of an infant formula is dictated by federal
regulations, which define macronutrient, vitamin, mineral, and
other ingredient levels in an effort to simulate the nutritional
and other properties of human breast milk. Infant formulas are
designed to support overall health and development in a pediatric
human subject, such as an infant or a child.
[0212] In some embodiments, the nutritional composition of the
present disclosure is a growing-up milk. Growing-up milks are
fortified milk-based beverages intended for children over 1 year of
age (typically from 1-3 years of age, from 4-6 years of age or from
1-6 years of age). They are not medical foods and are not intended
as a meal replacement or a supplement to address a particular
nutritional deficiency. Instead, growing-up milks are designed with
the intent to serve as a complement to a diverse diet to provide
additional insurance that a child achieves continual, daily intake
of all essential vitamins and minerals, macronutrients plus
additional functional dietary components, such as non-essential
nutrients that have purported health-promoting properties.
[0213] The exact composition of a growing-up milk or other
nutritional composition according to the present disclosure can
vary from market-to-market, depending on local regulations and
dietary intake information of the population of interest. In some
embodiments, nutritional compositions according to the disclosure
consist of a milk protein source, such as whole or skim milk, plus
added sugar and sweeteners to achieve desired sensory properties,
and added vitamins and minerals. The fat composition includes an
enriched lipid fraction derived from milk. Total protein can be
targeted to match that of human milk, cow milk or a lower value.
Total carbohydrate is usually targeted to provide as little added
sugar, such as sucrose or fructose, as possible to achieve an
acceptable taste. Typically, Vitamin A, calcium and Vitamin D are
added at levels to match the nutrient contribution of regional cow
milk. Otherwise, in some embodiments, vitamins and minerals can be
added at levels that provide approximately 20% of the dietary
reference intake (DRI) or 20% of the Daily Value (DV) per serving.
Moreover, nutrient values can vary between markets depending on the
identified nutritional needs of the intended population, raw
material contributions and regional regulations.
[0214] The disclosed nutritional composition(s) may be provided in
any form known in the art, such as a powder, a gel, a suspension, a
paste, a solid, a liquid, a liquid concentrate, a reconstituteable
powdered milk substitute or a ready-to-use product. The nutritional
composition may, in certain embodiments, comprise a nutritional
supplement, children's nutritional product, infant formula, human
milk fortifier, growing-up milk or any other nutritional
composition designed for an infant or a pediatric subject.
Nutritional compositions of the present disclosure include, for
example, orally-ingestible, health-promoting substances including,
for example, foods, beverages, tablets, capsules and powders.
Moreover, the nutritional composition of the present disclosure may
be standardized to a specific caloric content, it may be provided
as a ready-to-use product, or it may be provided in a concentrated
form. In some embodiments, the nutritional composition is in powder
form with a particle size in the range of 5 .mu.m to 1500 .mu.m,
more preferably in the range of 10 .mu.m to 300 .mu.m.
[0215] The nutritional compositions of the present disclosure may
be provided in a suitable container system. For example,
non-limiting examples of suitable container systems include plastic
containers, metal containers, foil pouches, plastic pouches,
multi-layered pouches, and combinations thereof. In certain
embodiments, the nutritional composition may be a powdered
composition that is contained within a plastic container. In
certain other embodiments, the nutritional composition may be
contained within a plastic pouch located inside a plastic
container.
[0216] In some embodiments, the method is directed to manufacturing
a powdered nutritional composition. The term "powdered nutritional
composition" as used herein, unless otherwise specified, refers to
dry-blended powdered nutritional formulations comprising protein,
and specifically plant protein, and at least one of fat and
carbohydrate, which are reconstitutable with an aqueous liquid, and
which are suitable for oral administration to a human.
[0217] Indeed, in some embodiments, the method comprises the steps
of dry-blending selected nutritional powders of the nutrients
selected to create a base nutritional powder to which additional
selected ingredients, such as dietary butyrate, may be added and
further blended with the base nutritional powder. The term
"dry-blended" as used herein, unless otherwise specified, refers to
the mixing of components or ingredients to form a base nutritional
powder or, to the addition of a dry, powdered or granulated
component or ingredient to a base powder to form a powdered
nutritional formulation. In some embodiments, the base nutritional
powder is a milk-based nutritional powder. In some embodiments, the
base nutritional powder includes at least one fat, one protein, and
one carbohydrate. The powdered nutritional formulations may have a
caloric density tailored to the nutritional needs of the target
subject.
[0218] The powdered nutritional compositions may be formulated with
sufficient kinds and amounts of nutrients so as to provide a sole,
primary, or supplemental source of nutrition, or to provide a
specialized powdered nutritional formulation for use in individuals
afflicted with specific diseases or conditions. For example, in
some embodiments, the nutritional compositions disclosed herein may
be suitable for administration to pediatric subjects and infants in
order provide exemplary health benefits disclosed herein.
[0219] The powdered nutritional compositions provided herein may
further comprise other optional ingredients that may modify the
physical, chemical, hedonic or processing characteristics of the
products or serve as nutritional components when used in the
targeted population. Many such optional ingredients are known or
otherwise suitable for use in other nutritional products and may
also be used in the powdered nutritional compositions described
herein, provided that such optional ingredients are safe and
effective for oral administration and are compatible with the
essential and other ingredients in the selected product form.
Non-limiting examples of such optional ingredients include
preservatives, antioxidants, emulsifying agents, buffers,
additional nutrients as described herein, colorants, flavors,
thickening agents and stabilizers, and so forth.
[0220] The powdered nutritional compositions of the present
disclosure may be packaged and sealed in single or multi-use
containers, and then stored under ambient conditions for up to
about 36 months or longer, more typically from about 12 to about 24
months. For multi-use containers, these packages can be opened and
then covered for repeated use by the ultimate user, provided that
the covered package is then stored under ambient conditions (e.g.,
avoid extreme temperatures) and the contents used within about one
month or so.
[0221] In some embodiments, the method further comprises the step
of placing the nutritional compositions in a suitable package. A
suitable package may comprise a container, tub, pouch, sachet,
bottle, or any other container known and used in the art for
containing nutritional composition. In some embodiments, the
package containing the nutritional composition is a plastic
container. In some embodiments, the package containing the
nutritional composition is a metal, glass, coated or laminated
cardboard or paper container. Generally, these types of packaging
materials are suitable for use with certain sterilization methods
utilized during the manufacturing of nutritional compositions
formulated for oral administration.
[0222] In some embodiments, the nutritional compositions are
packaged in a container. The container for use herein may include
any container suitable for use with powdered and/or liquid
nutritional products that is also capable of withstanding aseptic
processing conditions (e.g., sterilization) as described herein and
known to those of ordinary skill in the art. A suitable container
may be a single-dose container, or may be a multi-dose resealable,
or reclosable container that may or may not have a sealing member,
such as a thin foil sealing member located below the cap.
Non-limiting examples of such containers include bags, plastic
bottles or containers, pouches, metal cans, glass bottles, juice
box-type containers, foil pouches, plastic bags sold in boxes, or
any other container meeting the above-described criteria. In some
embodiments, the container is a resealable multi-dose plastic
container. In certain embodiments, the resealable multi-dose
plastic container further comprises a foil seal and a plastic
resealable cap. In some embodiments, the container may include a
direct seal screw cap. In other embodiments, the container may be a
flexible pouch.
[0223] In some embodiments, the nutritional composition is a liquid
nutritional composition and is processed via a "retort packaging"
or "retort sterilizing" process. The terms "retort packaging" and
"retort sterilizing" are used interchangeably herein, and unless
otherwise specified, refer to the common practice of filling a
container, most typically a metal can or other similar package,
with a nutritional liquid and then subjecting the liquid-filled
package to the necessary heat sterilization step, to form a
sterilized, retort packaged, nutritional liquid product.
[0224] In some embodiments, the nutritional compositions disclosed
herein are processed via an acceptable aseptic packaging method.
The term "aseptic packaging" as used herein, unless otherwise
specified, refers to the manufacture of a packaged product without
reliance upon the above-described retort packaging step, wherein
the nutritional liquid and package are sterilized separately prior
to filling, and then are combined under sterilized or aseptic
processing conditions to form a sterilized, aseptically packaged,
nutritional liquid product.
[0225] The nutritional compositions described herein, in some
embodiments, advantageously promote healthy weight maintenance in
target subject and reduce the incidence of obesity and metabolic
syndrome in target subjects. Indeed, early nutrition is important
for healthy metabolic programming. Healthy weight and metabolism
require a tight interplay between organs. Without being bound by
any particular theory, dietary butyrate is thought to assist with
early cellular and tissue programming that provides healthy body
composition and metabolism and of associated tissues, such as brain
tissue. Further, provided are methods for improving adipose tissue
functioning and/or improving the quality of adipose tissue in
target subjects. Further provided are methods for reducing the
incidence of obesity and metabolic syndrome in target subjects.
Also provided are methods for supporting, reinforcing, or improving
healthy weight and metabolism in target subjects who do not have a
healthy weight or metabolism. In some embodiments, the target
subject is a formula-fed infant or a pediatric subject that was
formula-fed.
[0226] In some embodiments, the method comprises the step of
administering the nutritional composition disclosed herein
comprising dietary butyrate to the target subject. Indeed, in
certain embodiments where the target subject is a formula-fed
infant, the formula-fed infant will experience an improvement in
adipose tissue function and quality as compared to other
formula-fed infants that are not provided the nutritional
composition including dietary butyrate.
[0227] In some embodiments the target subject may be a pediatric
subject. Further, in one embodiment, the nutritional composition
provided to the pediatric subject may be an infant formula. In
certain embodiment, the dietary butyrate may be formulated in an
infant formula together with other ingredients, such as DHA, ARA,
lactoferrin, PE, sphingomyelin, inositol, ALA, EGCG, sulforaphane,
vitamin D, and combinations thereof. Without being bound by any
particular theory the combination of dietary butyrate together with
these selected ingredients may act synergistically and provide
synergistic health benefits to the target subject.
[0228] Further provided are methods of a staged infant feeding
regimen to promote healthy development and growth, such as
increased and/or accelerated myelination, in a formula-fed infant.
In certain embodiments, the feeding regimen includes feeding a
first nutritional composition, a second nutritional composition,
and a third nutritional composition to an infant as the infant
ages. In the respective staged feeding regimen disclosed herein,
each of the first, second, third, etc. nutritional compositions are
different from each other.
[0229] Indeed, while a conventional "one size fits all" infant
formula can provide adequate nutrition for a formula-fed infant,
such formulas do not account for the changing requirements during
development. Accordingly, it would be beneficial to provide an
infant feeding regimen which includes nutritional compositions
tailored to provide a combination of nutrients designed to promote
healthy development and growth at each stage. Furthermore, it is
beneficial to provide staged nutritional compositions tailored to
continue promoting and accelerating myelination in a formula-fed
infant throughout the first 12 months of life.
[0230] "Infant" is a human from birth through not more than 12
months of age, where a "newborn infant" is an infant from birth
through 3 months of age, a "middle stage infant" is an infant from
3 months of age through 6 months of age, and a "later stage infant"
is an infant from 6 months of age through 12 months of age or 1
year.
[0231] Accordingly, in certain embodiments, the first nutritional
composition is fed to an infant that is a newborn infant, i.e. age
birth through 3 months of age, the second nutritional composition
is fed to a middle stage infant, i.e. an infant that is age 3
months of age to 6 months of age, and the third composition is fed
to a later stage infant, i.e. an infant that is age 6 months of age
to 12 months of age.
[0232] In certain embodiments, the feeding regimen of the present
disclosure includes:
A) a first nutritional composition comprising:
[0233] i) up to about 0.2 mg/100 kcal of iron;
[0234] ii) up to about 0.7 mg/100 kcal of zinc;
[0235] iii) up to about 2 g/100 kcal of protein;
[0236] iv) a whey:casein ratio of at least 72:28 and, in
embodiments, between 72:28 and 90:10;
[0237] v) up to 35 mg/100 kcal of inositol;
[0238] vi) up to 9.0 mg/100 kcal of butyrate; and/or
[0239] vii) from about 1.8 .mu.g/100 kcal to about 2.0 .mu.g/100
kcal of vitamin D;
B) a second nutritional composition comprising:
[0240] i) from about 0.3 mg/100 kcal to about 0.6 mg/100 kcal of
iron;
[0241] ii) up to about 0.9 mg/100 kcal of zinc;
[0242] iii) up to about 1.9 g/100 kcal of protein;
[0243] iv) a whey:casein ratio of between 56:44 and 68:32;
[0244] v) up to 25 mg/100 kcal of inositol;
[0245] vi) up to 9.0 mg/100 kcal of butyrate; and/or
[0246] vii) from about 1.8 .mu.g/100 kcal to about 2.0 .mu.g/100
kcal of vitamin D; and
C) a third nutritional composition comprising:
[0247] i) from about 0.3 mg/100 kcal to about 1.2 mg/100 kcal of
iron;
[0248] ii) up to about 1 mg/100 kcal of zinc;
[0249] iii) up to about 1.5 g/100 kcal of protein;
[0250] iv) a whey:casein ratio of between 45:55 and 68:32;
[0251] v) up to 20 mg/100 kcal of inositol;
[0252] vi) up to 9.0 mg/100 kcal of butyrate; and/or
[0253] vii) from about 1.8 .mu.g/100 kcal to about 2.0 .mu.g/100
kcal of vitamin D.
[0254] In certain embodiments, the feeding regimen of the present
disclosure includes:
A) a first nutritional composition comprising:
[0255] i) up to about 0.45 mg/100 kcal of iron;
[0256] ii) up to about 0.7 mg/100 kcal of zinc;
[0257] iii) up to about 2 g/100 kcal of protein;
[0258] iv) a whey:casein ratio of at least 72:28 and, in
embodiments, between 72:28 and 90:10;
[0259] v) up to 35 mg/100 kcal of inositol;
[0260] vi) up to 9.0 mg/100 kcal of butyrate; and/or
[0261] vii) from about 1.8 .mu.g/100 kcal to about 2.0 .mu.g/100
kcal of vitamin D;
B) a second nutritional composition comprising:
[0262] i) from about 0.3 mg/100 kcal to about 0.6 mg/100 kcal of
iron;
[0263] ii) up to about 0.9 mg/100 kcal of zinc;
[0264] iii) up to about 1.9 g/100 kcal of protein;
[0265] iv) a whey:casein ratio of between 56:44 and 68:32;
[0266] v) up to 25 mg/100 kcal of inositol;
[0267] vi) up to 9.0 mg/100 kcal of butyrate; and/or
[0268] vii) from about 1.8 .mu.g/100 kcal to about 2.0 .mu.g/100
kcal of vitamin D; and
C) a third nutritional composition comprising:
[0269] i) from about 0.6 mg/100 kcal to about 1.2 mg/100 kcal of
iron;
[0270] ii) up to about 2 mg/100 kcal of zinc;
[0271] iii) up to about 3 g/100 kcal of protein;
[0272] iv) a whey:casein ratio of between 45:55 and 54:46;
[0273] v) up to 20 mg/100 kcal of inositol;
[0274] vi) up to 9.0 mg/100 kcal of butyrate; and/or
[0275] vii) from about 1.8 .mu.g/100 kcal to about 2.0 .mu.g/100
kcal of vitamin D.
[0276] In certain embodiments, the feeding regimen of the present
disclosure includes:
[0277] A) a first nutritional composition comprising: [0278] i) no
greater than 9.0 mg/100 kcal of butyrate; and
[0279] B) a second nutritional composition comprising: [0280] i) at
least 9.0 mg/100 kcal of butyrate. Indeed, in this example
embodiment the level of butyrate is increased from the first
composition compared to the second composition. Indeed, further,
the first composition may be provided to an infant from the age of
birth to three months and the second composition is then fed to the
infant when the infant is age 3 months to 12 months. Indeed, the
level of butyrate is increased as the infant increases in age.
Further, in some embodiments the first composition comprises other
nutrients such as sialic acid, human milk oligosaccharides, and/or
lactoferrin. Indeed, In some embodiments the levels of these
additional nutrients are actually higher in the first composition
as compared to the second composition. Accordingly, while the
concentration of dietary supplied butyrate is increased as the
infant ages, the concentration of these other nutrients is
decreased as the infant ages.
[0281] In certain embodiments, the feeding regimen of the present
disclosure includes: A) a first nutritional composition comprising
from about 7 .mu.g/mL to about 22 .mu.g/mL of butyrate; B) a second
nutritional composition comprising from about 17 .mu.g/mL to about
24 .mu.g/mL of butyrate; and C) a third nutritional composition
comprising from about 17 .mu.g/mL to about 22 .mu.g/mL of
butyrate.
[0282] In certain embodiments, the feeding regimen of the present
disclosure includes: A) a first nutritional composition comprising
from about 5 .mu.g/mL to about 30 .mu.g/mL of butyrate; B) a second
nutritional composition comprising from about 15 .mu.g/mL to about
25 .mu.g/mL of butyrate; and C) a third nutritional composition
comprising from about 17 .mu.g/mL to about 22 .mu.g/mL of
butyrate.
[0283] Indeed, in some embodiments the first nutritional
composition is administered to an infant from birth to the age of 6
months, the second nutritional composition is administered to an
infant from 6 months to the age of 12 months, and the third
nutritional composition is administered to an infant/child after
the age of 12 months.
[0284] Also provided is a staged feeding regimen for an infant or
child where the infant is administered a series of nutritional
compositions comprising
[0285] A) a first nutritional composition comprising from about 7
.mu.g/mL to about 10 .mu.g/mL of dietary butyrate that is
administered to the infant from birth to the age of two weeks;
[0286] B) a second nutritional composition comprising from about 9
.mu.g/mL to about 13 .mu.g/mL of dietary butyrate that is
administered to the infant from the age of two weeks to 4
weeks;
[0287] C) a third nutritional composition comprising from about 16
.mu.g/mL to about 22 .mu.g/mL of dietary butyrate that is
administered to the infant from the age of 4 weeks to 13 weeks;
[0288] D) a fourth nutritional composition comprising from about 17
.mu.g/mL to about 24 .mu.g/mL of dietary butyrate that is
administered to the infant from the age of 13 weeks to about 26
weeks; and
[0289] E) a fifth nutritional composition comprising from about 17
.mu.g/mL to about 22 .mu.g/mL of dietary butyrate that is
administered to the infant from the age of 26 weeks to about 52
weeks.
[0290] Indeed, in certain embodiments, the feeding regimen is
designed to provide the highest concentration of dietary butyrate
to the infant when the infant is at least 26 weeks old.
[0291] In certain embodiments, the first nutritional composition
may have a whey:casein ratio of about 80:20; in other embodiments,
the first nutritional composition may have a whey:casein ratio of
from about 72:28 to about 90:10.
[0292] In certain embodiments, the second nutritional composition
may have a whey:casein ratio of from about 60:40. In other
embodiments, the second nutritional composition may have a
whey:casein ratio of from about 56:44 to about 68:32. Still, in
certain embodiments, the second nutritional composition may have a
whey:casein ratio of from about 56:44 to about 70:30.
[0293] In certain embodiments, the third nutritional composition
may have a whey:casein ratio of from about 50:50. In other
embodiments, the third nutritional composition may have a
whey:casein ratio of from about 45:55 to about 54:46.
Example 2
[0294] This example illustrates an embodiment of the feeding
regimen of the present disclosure. Each nutritional composition
disclosed herein may include other nutrients provided herein that
are not listed in the Tables below. Provided below is an example of
a first nutritional composition.
TABLE-US-00005 Description UOM per 100 kcal Iron mg 0.2 Zinc mg 0.7
Protein g 2.0 Whey:Casein Ratio 80:20 Inositol mg 35 Butyrate mg
9.0 Vitamin D .mu.g 1.8-2.0
Provided below is an example of a second nutritional
composition.
TABLE-US-00006 Description UOM per 100 kcal Iron mg 0.3-0.6 Zinc mg
0.9 Protein g 1.9 Whey:Casein Ratio 60:40 Inositol mg 25 Butyrate
mg 9 Vitamin D .mu.g 1.8-2.0
Provided below is an example of a third nutritional
composition.
TABLE-US-00007 Description UOM per 100 kcal Iron mg 0.3-1.2 Zinc mg
3.0 Protein g 1.5 Whey:Casein Ratio 50:50 Inositol mg 20 Butyrate
mg 9.0 Vitamin D .mu.g 1.8-2.0
Example 3
[0295] This example illustrates an embodiment of the feeding
regimen of the present disclosure. Each nutritional composition
disclosed herein may include other nutrients provided herein that
are not listed in the Tables below. Provided below is an example of
a first nutritional composition.
TABLE-US-00008 Description UOM per 100 kcal Iron mg 0.46 Zinc mg
0.7 Protein g 2.0 Whey:Casein Ratio 80:20 Inositol mg 35 Butyrate
mg 9 Vitamin D .mu.g 1.8-2.0
Provided below is an example of a second nutritional
composition.
TABLE-US-00009 Description UOM per 100 kcal Iron mg 0.3-0.6 Zinc mg
0.9 Protein g 1.9 Whey:Casein Ratio 60:40 Inositol mg 25 Butyrate
mg 9 Vitamin D .mu.g 1.8-2.0
Provided below is an example of a third nutritional
composition.
TABLE-US-00010 Description UOM per 100 kcal Iron mg 0.6-1.2 Zinc mg
2 Protein g 3 Whey:Casein Ratio 50:50 Inositol mg 20 Butyrate mg 9
Vitamin D .mu.g 1.8-2.0
[0296] All combinations of method or process steps as used herein
can be performed in any order, unless otherwise specified or
clearly implied to the contrary by the context in which the
referenced combination is made.
[0297] The methods and compositions of the present disclosure,
including components thereof, can comprise, consist of, or consist
essentially of the essential elements and limitations of the
embodiments described herein, as well as any additional or optional
ingredients, components or limitations described herein or
otherwise useful in nutritional compositions.
Example 4
[0298] Example 4 illustrates a method for accurately determining
the level of butyrate present in samples of human breast milk.
Indeed, limited references are available discussing the levels of
butyrate found in human breast milk and often reported
concentration levels of butyrate in human breast milk differs
significantly from reports in literature. Also, butyrate can be
present in its free form as butyric acid or bound with a
triacylglycerol molecule. As such, it is also unclear from the
current literature if total butyrate is reported or if only the
free form of butyrate is reported.
[0299] The following Definitions may be used in Example 4.
TABLE-US-00011 TABLE 5 Definitions Term Definitions BA Butyric Acid
BA-D7 Butyric Acid (D7) deuterated IS Internal standard NPH.sub.2O
Nanopure water N/A Not available RT Room temperature CoA
Certificate of Analysis EtAc Ethyl Acetate H.sub.3PO.sub.4
Phosphoric acid CRL Lipase from Candida rugose PBS 0.1 Phosphate
Buffered Saline SSA 5-sulfosalicylic acid HM Human milk GC Gas
chromatography MSD Mass Spectrometer Detector EI Electron
ionization GC-MS Gas Chromatography combined with Mass Spectrometer
Min Minute O/N Overnight Ex Example
[0300] The following Equipment and Apparatus may be used in Example
4. Note, equivalents may be used unless otherwise specified.
TABLE-US-00012 TABLE 6 Equipment & Apparatus Equipment
Description (brand, make, model, etc) Gas Chromatography Agilent
7890B series GC Mass spectrometer Agilent 5977A inert EI Source MSD
Hydrogen generator Parker Hannifan Corp. H2PD-300 Autosampler
Agilent 7693A autoinjector and tray Analytical column Phenomenex
Zebron ZB-FFAP, 30 m .times. 0.5 mm .times. 1.00 .mu.m Balance
Mettler Toledo with anti-static kit or equivalent. Sensibility of
balance to .+-.0.1 mg Centrifuge Sorvall Biofuge
Stratos-Model#LR56495, Relabive force to 10,000 G Vortex mixer VWR
Analog vortex mixer (Model #VM-3000) or equivalent Thermo mixer C
Eppendorf Thermo Mixer C with Thermo Top Sonicator Fisher
SolidState/Ultrasonic FS-28 Pipettor Biohit Pipette tips Biohit
Optifit 10 .mu.L-1 mL or equivalent 1.5 mL screw cap tubes Sarstedt
#72692 1.5 mL polypropylene with screw cap, centrifugation to
20,000 .times. g 1.5 mL tubes Eppendorf Protein Lo-bind 1.5 mL
tubes #022431081 Autosampler vials Agilent 12 .times. 32 mm, clear
screw top GC 2 mL vials #5183-2067 Silicone septa screw caps
Agilent PTFE; silicone/PTFE septa screw cap #5185-5862 Glass insert
Agilent 250 .mu.L deactivated glass insert with polymer feet
#5181-8872 Stir plate Corning PC-351 Hotplate-Stirrer 10 .mu.L, 25
.mu.L, 50 .mu.L, 100 .mu.L Gas tight syringes
[0301] The following Chemicals and Reagents may be used in Example
4.
TABLE-US-00013 TABLE 7 Chemicals and Reagents No. Reagent Name
Concentration Preparation No. Brand Shelf life Storage 1 NP water
18.2 M.OMEGA./cm N/A Barnstead 1 month RT 2 BupH PBS 0.1 M 1 Thermo
#28372 2 weeks RT 3 IS BA-D7 Neat N/A Cambridge isotope 1 year
4.degree. C. DLM-1508-5 4 IS BA-D7 1 mg/mL in NPH.sub.2O 2 N/A
Daily RT 5 IS BA-D7 1 mg/mL in EtAc V.A-2 N/a Monthly -20.degree.
C. 6 CRL Powder N/A Sigma I8525-5 mu 1 year N/A 7 CRL 3 mg/mL 3 N/A
Daily N/A 8 BA Neat N/A Sigma 19215 1 year 2 9 BA V.A-1 V.A-1 N/A
Monthly 2 10 SSA Powder N/A Sigma S7422 3 years 3 11 SSA 1 mg/.mu.L
4 N/A Make daily N/A 12 o-phosphoric acid .gtoreq.85% N/A Fisher
Scientific, 3 years 4 (ACS grade) A242-500 13 EtAc 99.9% N/A Sigma
#650528 1 year 5 14 Innovative N/A N/A Innovative Research 10 years
6 human milk #IR1000042 15 Control- N/A (aliquot) 5.6 Innovative
Research 10 years 6 Innovative #IR1000042 human milk
[0302] Accordingly, based on multiple questions regarding total
butyrate levels in human milk and gaps found in the literature,
Example 4 was developed and utilizes a nonspecific microbial
triacylglycerol lipase form the yeast family to hydrolyze bound
butyrate to free butyric acid, which is then deproteinized,
acidified, and extracted into an organic solvent. The butyric acid
in the extract is separated and identified from other compounds on
a polar capillary column and analyzed by Gas Chromatography
combined with Mass Spectrometry (GC-MS). Due to the volatility of
butyric acid, and internal standard of deuterated butyric acid
(BA-D7) is used during quantification.
[0303] The human milk samples are prepared according the following
procedure. A 500 mL solution of 0.1M PBS was prepared according to
the following steps. Add 1 pouch of BupH PBS to a 500 mL bottle, QS
to 500 mL with NPH.sub.2O, add a stir bar, and mix on a stir plate
until the pouch contents are dissolved. The resulting solution is a
0.1 M PBS solution having a pH of 7.2 when the pouch contents are
dissolved in the final volume of 500 mL NPH.sub.2O.
[0304] An internal standard of BA-D7 was prepared according to the
following steps. Tare a 2 mL GC vial with screw cap, remove the cap
and add 1.5 .mu.L IS BA-D7 by pipette. Replace the cap and
accurately weigh the IS, record weight. Multiply weight by purity,
subtract the volume and re-suspend with NPH.sub.2O at 1 mg/mL.
[0305] A lipase from Candida rugosa (CRL) of 3 mg/mL in PBS was
prepared according to the following steps. First, the total amount
of lipase from Candida rugosa was calculated based on the total
number of samples and controls being prepared. For example, (10
samples+1 control).times.240 .mu.g lipase=2.64 mg of lipase. Also,
240 .mu.g of lipase was accounted for for each sample in order to
account for any pipetting loss. Once the amount of lipase needed is
determined, resuspend the lipase in PBS at 3 mg/mL. Next, weigh out
the total amount of lipase needed in a 1.5 mL protein Lo-bind
tubes, record the weight of the CRL. Store on ice until needed, Add
calculated volume of 0.1 MPBS, vortex, heat on Thermo mixer at 37
deg. C. at 900 RPM for 30 minutes to get into solution.
[0306] A 5-sulfosalicylic acid (SAA) 1 mg/.mu.L in water solution
was prepared according to the following steps. Calculated the total
amount of SSA needed for the number of samples and control being
prepared. For example, (10 samples+1 control).times.14 mg SSA=154
mg of SSA. Weigh out the total amount needed in a 1.5 mL protein
Lo-bind tube, record the SSA weight. Re-suspend the SSA with
NPH.sub.2O at 1 mg/.mu.L, for example 154 mg of SSA would be
re-suspended with 154 .mu.L of NPH.sub.2O.
[0307] Human milk samples were prepared according to the following
steps. Thaw a 50 mL container of Innovative Human milk over night
at 4 deg. C. with gentle rocking, vortex, and aliquot 0.750 mL into
pre-chilled 1.5 mL screw cap tubes. Vortex 50 mL container every
sixth aliquot to make sure milk control sample stays mixed, then
store in 1.5 mL tubes in -80 deg. C. Pull 2 mL of human milk
samples and control from -80 deg. C. thaw in 4 deg. C. for about
one hour, then put at room temperature until completely thawed.
Invert 10 times to mix and vortex.
[0308] The butyric acid standard was prepared according to the
following procedure. Make a 1 mg/mL solution of butyric acid
standard in ethyl acetate. Tare a 5 mL volumetric flask with glass
stopper, add 5.2 .mu.L butyric acid with a pipette, weight flask
and butyric acid, record butyric acid weight, QS flask to 5 mL,
invert 20 times to mix. Calculate mg/mL using purity from
Certificate of Analysis. For example, if the actual weight of
butyric acid is 5.04 mg, then actual weight 5.04 mg.times.99.06%
purity=5.02 mg/5 mL 1 mg/mL=1 .mu.g/.mu.L. Store in -20 deg. C. The
butyric acid standards of 1 mg/.mu.L need to be prepared
monthly.
[0309] An internal standard BA-D7 procedure is prepared according
to the following. Make a 1 mg/mL solution of the internal standard
BA-D7 in ethyl acetate. Tare a 5 mL volumetric flask with a gas
stopper, add 4.9 .mu.L BA-D7 with a pipette, weigh flask and BA-D7,
record BA-D7 weight. QS flask to 5 mL, invert 20 times to mix.
Calculate mg/mL using purity from the Certificate of Analysis. For
Example: actual weight 5.07 mg.times.98.5% purity=4.99 mg/5 mL,
1.00 mg/mL=1 .mu.g/.mu.L. Store in -20 deg. C. The internal
standard BA-D7 1 mg/mL needs to be prepared monthly.
TABLE-US-00014 TABLE 8 Preparation of working butyric acid +
internal standard BA-D7 calibration standard curve Calibration
points 1 2 3 4 5 Butyric acid 1 .mu.g 10 .mu.g 50 .mu.g 100 .mu.g
150 .mu.g amount needed IS BA-D7 15 .mu.g 15 .mu.g 15 .mu.g 15
.mu.g 15 .mu.g amount needed Ethyl QS 1 mL QS 1 mL QS 1 mL QS 1 mL
QS 1 mL Acetate
TABLE-US-00015 TABLE 9 Example preparation of working BA + IS BA-D7
calibration standard curve Calibration points 1 2 3 4 5 BA volume 1
.mu.L 10 .mu.L 50 .mu.L 100 .mu.L 150 .mu.L from 1 mg/mL IS BA-D7
15 .mu.L 15 .mu.L 15 .mu.L 15 .mu.L 15 .mu.L volume from 1 mg/mL
Ethyl 984 .mu.L 975 .mu.L 935 .mu.L 885 .mu.L 835 .mu.L acetate 1
mL
[0310] Prepare the above 5 point working calibration standard curve
in GC screw top vials, use gas tight syringes to add BA 1 mg/mL, IS
BA-D7 1 mg/mL and ethyl acetate. Vortex to mix and store in -20
deg. C. until needed. Note: working calibration standards need to
be prepared weekly.
[0311] Next, pull working calibration standards 1-5 from -20 deg.
C., allow standards to warm to room temperature (approximately
20-30 minutes), vortex to mix. Transfer 150 .mu.L to labeled GC
vial with glass insert (note working standard 3 requires 3
replicates, (1) calibration standard curve, (1) after sixth sample
to check for drift, and (1) at the end of the analyitcal run to
check for drift).
Sample Preparation--Human Milk
[0312] For each sample and control label a 1.5 mL screw cap tubes,
up to 11 samples and 1 control. Pipette 500 .mu.L human milk (HM)
samples and control into the labeled 1.5 mL screw cap tube. Add 7.5
.mu.L 1 mg/mL IS BA-D7 with gas tight syringe, vortex 10 sec. The
internal standard is being added at 15 .mu.g/mL, since the sample
volume of HM is 0.5 mL, 7.5 .mu.g is added. Sonicate the samples,
control and CRL for 5 minutes at room temperature. Add 240 .mu.g of
lipase. Pipette 80 .mu.L 3 mg/mL CRL solution into each sample and
control, vortex 10 sec., and place on Thermo mixer at 37 deg. C.
900 RPM for at least 17 hours. Note: if the samples are put on at 4
pm, they come off the Thermo mixer at 9:00 am. Pull samples and
control from the Thermo mixer, vortex and place on ice for 5
minutes. Add 14 .mu.L 1 mg/.mu.L SSA to each sample and control,
vortex 10 seconds and incubate at room temperature for 10 minutes.
Under a hood, add 30 .mu.L H.sub.3PO.sub.4 to each sample and
control, vortex 10 sec, then put on vortex mixer 5 minutes at 1300
RPM. Note: do not invert the 1.5 mL tubes after removing tubes from
vortex in order to prevent sample loss in the cap during the next
step. Under hood, add 500 .mu.L ethyl acetate to each sample and
control, vortex for 10 sec. then put on vortex mixer for 5 minutes
at 1300 RPM. Centrifuge at 14,000 RPM, which is equal to
18,188.times.g, for 45 minutes at room temperature. Transfer 200
.mu.L of top organic layer to labeled screw top GC vial with glass
insert. Store remaining sample in -20 deg. C. Put GC vials in
appropriate position on auto sampler.
Analysis of Samples
TABLE-US-00016 [0313] TABLE 10 Set up for Sample Analysis Step Task
1 Instrument Set-up: The GC-MS system should be located in an area
where temperature fluctuations will be minimal throughout the run.
2 Load the test method SCFA_MJN_MS.M and let the instruments
equilibrate for at least 20 minutes before running samples. 3 Set
up GC-MS instrument parameters according to the next table.
TABLE-US-00017 TABLE 11 Set-up for GC-MS Parameter Name Instrument
Set-up values Column Phenomenex Zebron ZB-FFAP, 30 m .times. 0.32
mm .times. 100 .mu.m Carry gas flow rate Hydrogen at 2.8 mL/min.
constant flow Injector temp. 250.degree. C. (200.degree. C. for
standby mode) Injection volume 1 .mu.L Injection mode Splitless
Transfer line 250.degree. C. (200.degree. C. for standby mode)
temperature Mass Source at 230.degree. and MS Quad at 150.degree.
spectrometer Temperature Rate Value Hold Run Program Initial
(.degree. C./min) (.degree. C.) time min time Min SCFA_MJN_MS.M
Initial 50 0 0 Ramp 1 20 120 0 3.5 Ramp 2 3 125 0 5.17 Ramp 3 1 130
0 10.17 Ramp 4 15 250 19 37.17 SCFA_Blank_MS.M Initial 70 0 0 Ramp
1 25 250 18 25.20 Standby_MS1column.M Initial 70 10 10.0
TABLE-US-00018 TABLE 12 Sample Sequence Set up Step Task 1 Sample
Sequence Set up: Once the GC-MS instrument is equilibrated, inject
a ''blank'' sample of ethyl acetate solution using SCFA_BLANK_MS.M
method followed by another blank of ethyl acetate solution using
method SCFA_MJN_MS.M. 2 Then inject each BA standard level (one
time) one after another and begin from low to high concentration
using method SCFA_MJN_MS.M. 3 Follow the last BA standard injection
with one blank of ethyl acetate solution injection using method
SCFA_Blank_MS.M. 4 Then inject 6 samples (one time) sequentially
using method SCFA_MJN_MS.M. 5 Follow each sample inject one blank
of ethyl acetate solution using method SCFA_Blank_MS.M. 6 Follow
the blank injection atfter the 6.sup.th sample or less run a middle
level of BA standard using method SCFA_MJN_MS.M. 7 Then inject a
blank of ethyl acetate solution at the end of the sequence. 8 If
more than 6 samples are to be run, a middle level of BA standard
have to be run again at the end of the sequence and then followed
by a solvent blank.
TABLE-US-00019 TABLE 13 End of Run Procedure Step Task 1 End of
Run: After all samples and standards have been analyzed, check the
last blank run. If needed, following the MS baking procedure to
bake the ionization source before loading the instrument standby
method. 2 The capillary column in the GC should always be flown
with carrier gas. 3 Turn the instrument from operation to standy
mode when the instrument is not in use by loading the method
Standby_MSl_column.M 4 If the instrument will be used in the next 2
hours, then keep the instrument in operational mode with method
SCFA_MJN_MS.M loaded.
TABLE-US-00020 TABLE 14 System Suitability Step Parameter 1 SYSTEM
SUITABILITY: Correlation coefficient R of standard curve should be
a minimum of 0.99. 2 Retention times must be within .+-.10% of
agreement between the standard and the samples. 3 Run an innovative
HM as check every time samples are run.
Calculation and Report
[0314] Follow the instrument calibration process to get butyric
acid result directly or use the following calculations to determine
the butyric acid (BA) concentration:
[0315] a. Use Microsoft Excel of qualified chromatography software
to plot a linear internal calibration curve using the ratio of
standard concentration to internal standards versus the ratio of
standard integration area to internal standards in the final
extracts. Display the regression trendline equation
(y=a*(C.sub.BA/C.sub.IS)+b) and R-squared value to the curve. The
R-squared value should be .gtoreq.0.990.
[0316] b. Calculate the butyric acid concentration (.mu.g/mL) in
the sample solution using linear regression trendline equation
displayed in the Excel calculation sheet from step Via, the sample
and extraction solvent volumes considered. Solve for the
C.sub.BA:
C BA = ( y - b ) * .times. C IS * V EtAc / ( a * .times. V sample )
##EQU00001##
[0317] C.sub.BA=Concentration of BA in the sample (.mu.g/mL)
[0318] y=Ratio of BA peak area to internal standards (IS) in the
injected sample solution
[0319] b=y-intercept of the curve
[0320] C.sub.IS=concentration of IS spiked into the sample
(.mu.g/mL)
[0321] V.sub.EtAc=Volume (.mu.L) of solvent ethyl acetate (EtAc)
used for extraction
[0322] a=slope of the curve
[0323] V.sub.sample=Sample volume (.mu.L)
[0324] c. The BA final results should be reported in (.mu.g/mL).
Use equation above to calculate results or get the results using
instrument software calibrations.
[0325] The blank and 5 point calibration standards can be loaded on
the autosampler and the analytical run started before beginning
sample extraction. In the sample sequence set up, there is a blank
between each sample to prevent carryover, and a standard and blank
after the sixth (6.sup.th) and last sample to check for drift. All
tips and 1.5 mL tubes used to analyze human milk are considered a
biological hazard and are autoclaved before disposal. Hydrogen
(H.sub.2) is flammable, be aware that H.sub.2 gas can flow into the
GC oven and create an explosion hazard. Always turn off the H.sub.2
supply before working on the instrument, ensure that the inlet and
detector column fittings are connected to a column or capped at all
times when H.sub.2 gas is supplied to the instrument.
[0326] IS BA-D7 is a combustible liquid, contact can cause severe
burns. Work in the fume hood or in a well-ventilated area and wear
safety gloves, lab coat and protective eye glasses. Buytric acid
(BA) is a combustible liquid, skin or eye contact can cause severe
burns. Work in the fume hood or in a well-ventilated area and wear
safety gloves, lab coat and protective eye glasses.
5-Sulfosalicylic acid (SSA) is a white powder to crystals, skin or
eye contact can cause severe burns. Wear safety gloves, lab coat
and protective eye glasses. Phosphoric acid 85% is corrosive and
has a strong pungent odor, and contact can cause severe burns. Work
in the fume hood or in a well-ventilated area and wear safety
gloves, lab coat and protective eye glasses. Ethyl Acetate is
highly flammable, it is harmful if inhaled or in contact with skin.
Wear protective clothing, gloves and eye glasses. Work in the fume
hood or in a well-ventilated area. Keep ethyl acetate away from
heat and open flame. When working with human milk (HM) wear safety
gloves, lab coat and protective eye glasses. Autoclave or bleach
all tips, tubes, and lab supplies that come into contact with
HM.
[0327] Method precision and accuracy: Precision: 1.8%-4.9% RSD;
Accuracy: 83.7%-90.2%. Limit of detection and working range: LOD:
0.7 .mu.g/mL; Range: 1 to 150 .mu.g/mL. General References used:
Method validation protocol 2016-001-MVP-AS.2002 vl and Report
2016-MVR-AS.2002 vl for the method AS.2002 vl: General Method for
Determination of Butyrate in Human Milk using Internal Standard by
GC-MS.
[0328] FIG. 1 illustrates an example chromatogram of ethyl acetate
solvent blank. FIG. 2 illustrates an example chromatogram of an
internal standard at 15 and butyric acid at 50 .mu.g/mL. FIG. 3
illustrates an example chromatogram of NP H.sub.2O blank through
the sample preparation. FIG. 4A illustrates a chromatogram of a
human milk sample and FIG. 4B illustrates the same chromatogram of
a human milk sample showing the internal standard and butyric acid
(BA) peaks. FIG. 5 is a sample sequence setup template. FIG. 6
illustrates a schematic process flow chart for the procedure
outlined in Example 4.
FORMULATION EXAMPLES
[0329] Formulation examples are provided to illustrate some
embodiments of the nutritional composition of the present
disclosure but should not be interpreted as any limitation thereon.
Other embodiments within the scope of the claims herein will be
apparent to one skilled in the art from the consideration of the
specification or practice of the nutritional composition or methods
disclosed herein. It is intended that the specification, together
with the example, be considered to be exemplary only, with the
scope and spirit of the disclosure being indicated by the claims
which follow the example.
[0330] Table 15 provides an example embodiment of a peptide
component including 8 peptides from Table 3.
TABLE-US-00021 TABLE 15 Example peptide component Example of
Selected Peptides for Peptide Component SEQ ID NO 5 SEQ ID NO 24
SEQ ID NO 33 SEQ ID NO 56 SEQ ID NO 64 SEQ ID NO 13 SEQ ID NO 24
SEQ ID NO 60
[0331] Table 16 provides an example embodiment of a peptide
component including certain peptides from Table 3.
TABLE-US-00022 TABLE 16 Example peptide component Example of
Selected Peptides for Peptide Component SEQ ID NO 13 SEQ ID NO 24
SEQ ID NO 60 SEQ ID NO 5 SEQ ID NO 11 SEQ ID NO 22 SEQ ID NO 25 SEQ
ID NO 33 SEQ ID NO 45 SEQ ID NO 46 SEQ ID NO 47 SEQ ID NO 48 SEQ ID
NO 52 SEQ ID NO 34 SEQ ID NO 36 SEQ ID NO 61 SEQ ID NO 62 SEQ ID NO
64
[0332] Table 17
[0333] Table 17, illustrated below, provides an example embodiment
of the nutritional profile of a nutritional composition including
dietary butyrate and describes the amount of each ingredient to be
included per 100 Kcal serving of nutritional composition.
TABLE-US-00023 TABLE 17 Nutrition profile of an example nutritional
composition including dietary butyrate per 100 Kcal Nutrient
Minimum Maximum Protein Equivalent Source (g) 1.0 7.0 Dietary
butyrate (mg) 0.5 300 Lactobacillus rhamnosus GG (cfu) 1 .times.
10.sup.4 1.5 .times. 10.sup.12 Carbohydrates (g) 6 22 Fat (g) 1.3
7.2 Prebiotic (g) 0.3 1.2 DHA (g) 4 22 Beta glucan (mg) 2.9 17
Probiotics (cfu) 0.5 5.0 Vitamin A (IU) 9.60 .times. 10.sup.5 3.80
.times. 10.sup.8 Vitamin D (IU) 134 921 Vitamin E (IU) 22 126
Vitamin K (mcg) 0.8 5.4 Thiamin (mcg) 2.9 18 Riboflavin (mcg) 63
328 Vitamin B6 (mcg) 68 420 Vitamin B12 (mcg) 52 397 Niacin (mcg)
0.2 0.9 Folic acid (mcg) 690 5881 Panthothenic acid (mcg) 8 66
Biotin (mcg) 232 1211 Vitamin C (mg) 1.4 5.5 Choline (mg) 4.9 24
Calcium (mg) 4.9 43 Phosphorus (mg) 68 297 Magnesium (mg) 54 210
Sodium (mg) 4.9 34 Potassium (mg) 24 88 Chloride (mg) 82 346 Iodine
(mcg) 53 237 Iron (mg) 8.9 79 Zinc (mg) 0.7 2.8 Manganese (mcg) 0.7
2.4 Copper (mcg) 7.2 41
[0334] All references cited in this specification, including
without limitation, all papers, publications, patents, patent
applications, presentations, texts, reports, manuscripts,
brochures, books, internet postings, journal articles, periodicals,
and the like, are hereby incorporated by reference into this
specification in their entireties. The discussion of the references
herein is intended merely to summarize the assertions made by their
authors and no admission is made that any reference constitutes
prior art. Applicants reserve the right to challenge the accuracy
and pertinence of the cited references.
[0335] Although embodiments of the disclosure have been described
using specific terms, devices, and methods, such description is for
illustrative purposes only. The words used are words of description
rather than of limitation. It is to be understood that changes and
variations may be made by those of ordinary skill in the art
without departing from the spirit or the scope of the present
disclosure, which is set forth in the following claims. In
addition, it should be understood that aspects of the various
embodiments may be interchanged in whole or in part. Therefore, the
spirit and scope of the appended claims should not be limited to
the description of the versions contained therein.
* * * * *